In addition, other questions can now be considered

In addition, other questions can now be considered. their antagonists inhibited the effect of E2. However, female macrophages failed to respond to E2 and managed elevated CD36, SR-A1 levels and lipid accumulation. FoxM1 inhibition in macrophages reduced ERs and enhanced CD36, SR-A1 expression, while FoxM1 overexpression in macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective role of M2 in female mice. M2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation. background showed reduced development of atherosclerotic lesions (26C27). The effect of the or mice with hypercholesterolemia resulted in a 30% reduction in the size of atherosclerotic lesions as well as macrophage content within the plaques (28). Unexpectedly, bone marrow transplantation experiments from your or WT mice into male mice did not reveal any role to M2 integrin in atherosclerosis (29). This inconsistency led us to further examine the role of M2 in atherosclerosis. Using and mice, we demonstrate a amazing, anti-atherogenic and gender-dependent role for M2 in hyperlipidemic female mice. Mechanistically, we find that M2 exerts this gender-specific effect by supporting of macrophage ER and ER expression and estrogen-dependent reduction of foam cell formation as a result of down-regulation of the lipid scavenger receptors CD36 and SR-A1. Materials and Methods Animals and Diet The mice in C57BL/6J background were from Jackson Laboratories (Bar Harbor, ME) and crossbred with mice to obtain littermate and mice (control mice designated as mice throughout). Both male and female mice were used in the experiments. Atherosclerosis was induced by placing 4-week-old and mice on a Western diet (High Fat Diet) made up of 0.2% cholesterol and 42% calories as fat (TD88137, Harlan Teklad) for 3 or 16 weeks. Control chow diet contained 18% protein and 5% excess fat (Teklad Global 2918, Harlan Teklad). All procedures were performed under protocols approved by the Cleveland Medical center IACUC. Reagents and antibodies Recombinant mouse GM-CSF and IL-4 were purchased from R&D Systems (Minneapolis, MN). The following antibodies were utilized for Western blot or FACS assays: mouse anti-CD36 (BD Biosciences, San Jose, CA), mouse anti-SRA-1 (R&D Systems), rabbit anti-SRB-1 (Thermoscientific), rat anti-LOX-1 and goat anti-CD206 (R&D Systems), rat anti-mouse ABCA-1 (Bio-Rad, Raleigh, NC), goat anti-ABCG-1and anti-Fox M1 (Santa Cruz, Dallas, TX), rabbit anti-PPAR, rabbit anti-ER, rabbit anti-ER (EMD Milipore, Temecula, CA) and mouse anti–actin and rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA). Mouse FITC-conjugated anti-M mAb (clone M1/70), PE or FITC-conjugated F4/80 mAb, anti-L-PE were from eBioscience (San Diego, CA). anti-X-PE, anti-4-PE mAbs were from (BD Biosciences, San Jose, CA). The anti-D antibody was provided by Dr. Yakubenko and was previously explained (27). Low-density lipoprotein/very-low density lipoprotein (LDL/VLDL) cholesterol was measured in mouse plasma using an HDL & LDL/VLDL Cholesterol Quantification Kit. Triglyceride levels were analyzed using Triglyceride Quantification Colorimetric/Fluorometric Kit (BioVision Research Products, Milpitas, CA). 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP), an ER antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-]pyrimidin-3-yl]phenol (PHTPP), a ER antagonist, were from Tocris Bioscience (Minneapolis, MN). Mouse IL-6, IL-12 p40/p70 and IL-10 Elisa packages were from RayBiotech (Norcross, GA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Modified LDL preparation LDL acetylation- LDL are combined with equal volume of saturated sodium acetate, mixed and cooled on ice. While constantly and slowly combining the solution, the volume of the required acetic anhydride was added in 3 actions over 20 minute intervals. The altered LDL are then dialyzed immediately in the chilly room against 100 fold excess of 09% NaCl, 0.05 EDTA, pH 7.4. The preparation is usually filtered through a 0.45 m filter. The protein content is determined by Lowry method and the extent of lysine modification is determined using TNBS assay (2,4,6-trinitrobenzene sulfonic acid). The protein content Opn5 of the acetylated LDL was 1mg/ml and the amount of altered lysine was ~58%. Each lot was 97% real by agarose gel electrophoresis. LDL oxidation-LDL was oxidized by incubating LDL with 5 M CuSO4 in phosphate-buffered saline without EDTA for 20h at 37C. Oxidation was arrested by refrigeration and addition of 100 M EDTA and 20 M butylated.The size of aortic root lesions was 3C4.5-fold larger in female than in mice. mice due to enhanced proliferation. M2 eradication advertised gender-dependent foam cell development due to improved uptake of cholesterol by macrophages. This difference was related to improved manifestation of lipid uptake receptors, Compact disc36 and scavenger receptor A1 (SR-A1), in feminine mice. Macrophages from feminine mice showed significantly reduced manifestation of FoxM1 transcription element and estrogen receptors (ER) and . 17-estradiol (E2) reduced Compact disc36, SR-A1 foam and amounts cell development in macrophages in ERC and ER-dependent way, as their antagonists inhibited the result of E2. Nevertheless, female macrophages didn’t react to E2 and taken care of elevated Compact disc36, SR-A1 amounts and lipid build up. FoxM1 inhibition in macrophages decreased ERs and improved Compact disc36, SR-A1 manifestation, while FoxM1 overexpression in macrophages reversed their proatherogenic phenotype. We demonstrate a fresh, surprising atheroprotective part of M2 in feminine mice. M2 maintains ER manifestation in macrophages and E2-reliant inhibition of foam cell development. background showed decreased advancement of atherosclerotic lesions (26C27). The result from the or mice with hypercholesterolemia led to a 30% decrease in how big is atherosclerotic lesions aswell as macrophage content material inside the plaques (28). Unexpectedly, bone tissue marrow transplantation tests through the or WT mice into man mice didn’t reveal any part to M2 integrin in atherosclerosis (29). This inconsistency led us to help expand examine the part of M2 in atherosclerosis. Using and mice, we demonstrate a unexpected, anti-atherogenic and gender-dependent part for M2 in hyperlipidemic feminine mice. Mechanistically, we discover that M2 exerts this gender-specific impact by assisting of macrophage ER and ER manifestation and estrogen-dependent reduced amount of foam cell development due to down-regulation from the lipid scavenger receptors Compact disc36 and SR-A1. Components and Methods Pets and Diet plan The mice in C57BL/6J history had been from Jackson Laboratories (Pub Harbor, Me personally) and crossbred with mice to acquire littermate and mice (control mice specified as mice throughout). Both male and feminine mice were found in the tests. Atherosclerosis was induced by putting 4-week-old and mice on the Traditional western diet plan (FAT RICH DIET) including 0.2% cholesterol and 42% calorie consumption as body fat (TD88137, Harlan Teklad) for 3 or 16 weeks. Control chow diet plan contained 18% proteins and 5% fats (Teklad Global 2918, Harlan Teklad). All methods had been performed under protocols authorized by the Cleveland Center IACUC. Reagents and antibodies Recombinant mouse GM-CSF and IL-4 had been bought from R&D Systems (Minneapolis, MN). The next antibodies were useful for Traditional western blot or FACS assays: mouse anti-CD36 (BD Biosciences, San Jose, CA), mouse anti-SRA-1 (R&D Systems), rabbit anti-SRB-1 (Thermoscientific), rat anti-LOX-1 and goat anti-CD206 (R&D Systems), rat anti-mouse ABCA-1 (Bio-Rad, Raleigh, NC), goat anti-ABCG-1and anti-Fox M1 (Santa Cruz, Dallas, TX), rabbit anti-PPAR, rabbit anti-ER, rabbit anti-ER (EMD Milipore, Temecula, CA) and mouse anti–actin and rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA). Mouse FITC-conjugated anti-M mAb (clone M1/70), PE or FITC-conjugated F4/80 mAb, anti-L-PE had been from eBioscience (NORTH PARK, CA). anti-X-PE, anti-4-PE mAbs had been from (BD Biosciences, San Jose, CA). The anti-D antibody was supplied by Dr. Yakubenko and once was referred to (27). Low-density lipoprotein/very-low denseness lipoprotein (LDL/VLDL) cholesterol was assessed in mouse plasma using an HDL & LDL/VLDL Cholesterol Quantification Package. Triglyceride levels had been examined using Triglyceride Quantification Colorimetric/Fluorometric Package (BioVision Research Items, Milpitas, CA). 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP), an ER antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-]pyrimidin-3-yl]phenol (PHTPP), a ER antagonist, had been from Tocris Bioscience (Minneapolis, MN). Mouse IL-6, IL-12 p40/p70 and IL-10 Elisa products had been from RayBiotech (Norcross, GA). All the reagents were bought from Sigma-Aldrich (St. Louis, MO). Modified LDL planning LDL acetylation- LDL are coupled with equal level of saturated sodium acetate, combined and cooled on snow. While consistently and slowly blending the solution, the quantity of the mandatory acetic anhydride was added in 3 measures over 20 minute intervals. The customized LDL are after that dialyzed over night in the cool space against 100 fold more than 09% NaCl, 0.05 EDTA, pH 7.4. The planning can be filtered through a 0.45 m filter. The proteins content depends upon Lowry method as well as the degree of lysine changes is set using TNBS assay (2,4,6-trinitrobenzene sulfonic acidity). The proteins content of the acetylated LDL was 1mg/ml and the amount of revised lysine was ~58%. Each lot was 97% genuine by agarose gel electrophoresis. LDL oxidation-LDL was oxidized by incubating LDL with 5 M CuSO4 in phosphate-buffered saline without EDTA for 20h at 37C. Oxidation was caught by refrigeration and addition of 100 M EDTA and 20 M butylated hydroxytoluene. Control incubations were carried out without CuS04 and with EDTA and butylated hydroxytoluene added prior to incubation (31). Protein content was determined by Lowry assay and the degree of oxidation of LDL was.Third, crossing mice into background did not switch expression levels of the tested integrins and HFD diet did not affect expression of the 2-integrins within the macrophages of both genders. macrophages in ERC and ER-dependent manner, as their antagonists inhibited the effect of E2. However, female macrophages failed to respond to E2 and managed elevated CD36, SR-A1 levels and lipid build up. FoxM1 inhibition in macrophages reduced ERs and enhanced CD36, SR-A1 manifestation, while FoxM1 overexpression in macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective part of M2 in female mice. M2 maintains ER manifestation in macrophages and E2-dependent inhibition of foam cell formation. background showed reduced development of atherosclerotic lesions (26C27). The effect of the or mice with hypercholesterolemia resulted in a 30% reduction in the size of atherosclerotic lesions as well as macrophage content within the plaques (28). Unexpectedly, bone marrow transplantation experiments from your or WT mice into male mice did not reveal any part to M2 integrin in atherosclerosis (29). This inconsistency led us to further examine the part of M2 in atherosclerosis. Using and mice, we demonstrate a amazing, anti-atherogenic and gender-dependent part for M2 in hyperlipidemic female mice. Mechanistically, we find that M2 exerts this gender-specific effect by assisting of macrophage ER and ER manifestation and estrogen-dependent reduction of foam cell formation as a result of down-regulation of the lipid scavenger receptors CD36 and SR-A1. Materials and Methods Animals and Diet The mice in C57BL/6J background were from Jackson Laboratories (Pub Harbor, ME) and crossbred with mice to obtain littermate and mice (control mice designated as mice throughout). Both male and female mice Lofexidine were used in the experiments. Atherosclerosis was induced by placing 4-week-old and mice on a Western diet (High Fat Diet) comprising 0.2% cholesterol and 42% calories as fat (TD88137, Harlan Teklad) for 3 or 16 weeks. Control chow diet contained 18% protein and 5% extra fat (Teklad Global 2918, Harlan Teklad). All methods were performed under protocols authorized by the Cleveland Medical center IACUC. Reagents and antibodies Recombinant mouse GM-CSF and IL-4 were purchased from R&D Systems (Minneapolis, MN). The following antibodies were utilized for Western blot or FACS assays: mouse anti-CD36 (BD Biosciences, San Jose, CA), mouse anti-SRA-1 (R&D Systems), rabbit anti-SRB-1 (Thermoscientific), rat anti-LOX-1 and goat anti-CD206 (R&D Systems), rat anti-mouse ABCA-1 (Bio-Rad, Raleigh, NC), goat anti-ABCG-1and anti-Fox M1 (Santa Cruz, Dallas, TX), rabbit anti-PPAR, rabbit anti-ER, rabbit anti-ER (EMD Milipore, Temecula, CA) and mouse anti–actin and rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA). Mouse FITC-conjugated anti-M mAb (clone M1/70), PE or FITC-conjugated F4/80 mAb, anti-L-PE were from eBioscience (San Diego, CA). anti-X-PE, anti-4-PE mAbs were from (BD Biosciences, San Jose, CA). The anti-D antibody was provided by Dr. Yakubenko and was previously explained (27). Low-density lipoprotein/very-low denseness lipoprotein (LDL/VLDL) cholesterol was measured in mouse plasma using an HDL & LDL/VLDL Cholesterol Quantification Kit. Triglyceride levels were analyzed using Triglyceride Quantification Colorimetric/Fluorometric Kit (BioVision Research Products, Milpitas, CA). 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP), an ER antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-]pyrimidin-3-yl]phenol (PHTPP), a ER antagonist, were from Tocris Bioscience (Minneapolis, MN). Mouse IL-6, IL-12 p40/p70 and IL-10 Elisa packages were from RayBiotech (Norcross, GA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Modified LDL preparation LDL acetylation- LDL are combined with equal volume of saturated sodium acetate, combined and cooled on snow. While continually and slowly combining the solution, the volume of the required acetic anhydride was added in 3 methods over 20 minute intervals. The revised LDL are then dialyzed immediately in.Images are representative of four indie experiments. macrophages. This difference was attributed to enhanced manifestation of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female mice showed dramatically reduced manifestation of FoxM1 transcription element and estrogen receptors (ER) and . 17-estradiol (E2) decreased CD36, SR-A1 levels and foam cell formation in macrophages in ERC and ER-dependent manner, as their antagonists inhibited the effect of E2. However, female macrophages failed to respond to E2 and managed elevated CD36, SR-A1 levels and lipid build up. FoxM1 inhibition in macrophages reduced ERs and enhanced Compact disc36, SR-A1 appearance, while FoxM1 overexpression in macrophages reversed their proatherogenic phenotype. We demonstrate a fresh, surprising atheroprotective function of M2 in feminine mice. M2 maintains ER appearance in macrophages and E2-reliant inhibition of foam cell development. background showed decreased advancement of atherosclerotic lesions (26C27). The result from the or mice with hypercholesterolemia led to a 30% decrease in how big is atherosclerotic lesions aswell as macrophage content material inside the plaques (28). Unexpectedly, bone tissue marrow transplantation tests in the or WT mice into man mice didn’t reveal any function to M2 integrin in atherosclerosis (29). This inconsistency led us to help expand examine the function of M2 in atherosclerosis. Using and mice, we demonstrate a astonishing, anti-atherogenic and gender-dependent function for M2 in hyperlipidemic feminine mice. Mechanistically, we discover that M2 exerts this gender-specific impact by helping of macrophage ER and ER appearance and estrogen-dependent reduced amount of foam cell development due to down-regulation from the lipid scavenger receptors Compact disc36 and SR-A1. Components and Methods Pets and Diet plan The mice in C57BL/6J history had been from Jackson Laboratories (Club Harbor, Me personally) and crossbred with mice to acquire littermate and mice (control mice specified as mice throughout). Both male and feminine mice were found in the tests. Atherosclerosis was induced by putting 4-week-old and mice on the Traditional western diet plan (FAT RICH DIET) filled with 0.2% cholesterol and 42% calorie consumption as body fat (TD88137, Harlan Teklad) for 3 or 16 weeks. Control chow diet plan contained 18% proteins and 5% unwanted fat (Teklad Global 2918, Harlan Teklad). All techniques had been performed under protocols accepted by the Cleveland Medical clinic IACUC. Reagents and antibodies Recombinant mouse GM-CSF and IL-4 had been bought from R&D Systems (Minneapolis, MN). The next antibodies were employed for Traditional western blot or FACS assays: mouse anti-CD36 (BD Biosciences, San Jose, CA), mouse anti-SRA-1 (R&D Systems), rabbit anti-SRB-1 (Thermoscientific), rat anti-LOX-1 and goat anti-CD206 (R&D Systems), rat anti-mouse ABCA-1 (Bio-Rad, Raleigh, NC), goat anti-ABCG-1and anti-Fox M1 (Santa Cruz, Dallas, TX), rabbit anti-PPAR, rabbit anti-ER, rabbit anti-ER (EMD Milipore, Temecula, CA) and mouse anti–actin and rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA). Mouse FITC-conjugated anti-M mAb (clone M1/70), PE or FITC-conjugated F4/80 mAb, anti-L-PE had been from eBioscience (NORTH PARK, CA). anti-X-PE, anti-4-PE mAbs had been from (BD Biosciences, San Jose, CA). The anti-D antibody was supplied by Dr. Yakubenko and once was defined (27). Low-density lipoprotein/very-low thickness lipoprotein (LDL/VLDL) cholesterol was assessed in mouse plasma using an HDL & LDL/VLDL Cholesterol Quantification Package. Triglyceride levels had been examined using Triglyceride Quantification Colorimetric/Fluorometric Package (BioVision Research Items, Milpitas, CA). 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP), an ER antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-]pyrimidin-3-yl]phenol (PHTPP), a ER antagonist, had been from Tocris Bioscience (Minneapolis, MN). Mouse IL-6, IL-12 p40/p70 and IL-10 Elisa sets had been from RayBiotech (Norcross, GA). All the reagents were bought from Sigma-Aldrich (St. Louis, MO). Modified LDL planning LDL acetylation- LDL are coupled with equal level of saturated sodium acetate, blended and cooled on glaciers. While frequently and slowly mixing up the solution, the quantity of the mandatory acetic anhydride was added in 3 techniques over 20 minute intervals. The improved LDL are after that dialyzed right away in the frosty area against 100 fold more than 09% NaCl, 0.05 EDTA, pH 7.4..Taken jointly, we recognize Fox M1 transcription matter as an integral enhancer of ERs expression in macrophages and indirect (via ERs) regulator of CD36, SR-A1 foam and levels cell formation. Open in another window Figure 8 Fox M1 regulates appearance of ERs in macrophages which is reduced exclusively in feminine macrophages(A) American blot evaluation of peritoneal macrophages produced from and mice given Compact disc or HFD probed with Stomach to FoxM1 and -actin. receptor A1 (SR-A1), in feminine mice. Macrophages from feminine mice showed significantly reduced appearance of FoxM1 transcription aspect and estrogen receptors (ER) and . 17-estradiol (E2) reduced Compact disc36, SR-A1 amounts and foam cell development in macrophages in ERC and ER-dependent way, as their antagonists inhibited the result of E2. Nevertheless, female macrophages didn’t react to E2 and preserved elevated Compact disc36, SR-A1 amounts and lipid deposition. FoxM1 inhibition in macrophages decreased ERs and improved Compact disc36, SR-A1 appearance, while FoxM1 overexpression in macrophages reversed their proatherogenic phenotype. We demonstrate a fresh, surprising atheroprotective function of M2 in feminine mice. M2 maintains ER appearance in macrophages and E2-reliant inhibition of foam cell formation. background showed reduced development of atherosclerotic lesions (26C27). The effect of the or mice with hypercholesterolemia resulted in a 30% reduction in the size of atherosclerotic lesions as well as macrophage content within the plaques Lofexidine (28). Unexpectedly, bone marrow transplantation experiments from the or WT Lofexidine mice into male mice did not reveal any role to M2 integrin in atherosclerosis (29). This inconsistency led us to further examine the role of M2 in atherosclerosis. Using and mice, we demonstrate a surprising, anti-atherogenic and gender-dependent role for M2 in hyperlipidemic female mice. Mechanistically, we find that M2 exerts this gender-specific effect by supporting of macrophage ER and ER expression and estrogen-dependent reduction of foam cell formation as a result of down-regulation of the lipid scavenger receptors CD36 and SR-A1. Materials and Methods Animals and Diet The mice in C57BL/6J background were from Jackson Laboratories (Bar Harbor, ME) and crossbred with mice to obtain littermate and mice (control mice designated as mice throughout). Both male and female mice were used in the experiments. Atherosclerosis was induced by placing 4-week-old and mice on a Western diet (High Fat Diet) made up of 0.2% cholesterol and 42% calories as fat (TD88137, Harlan Teklad) for 3 or 16 weeks. Control chow diet contained 18% protein and 5% fat (Teklad Global 2918, Harlan Teklad). All procedures were performed under protocols approved by the Cleveland Clinic IACUC. Reagents and antibodies Recombinant mouse GM-CSF and IL-4 were purchased from R&D Systems (Minneapolis, MN). The following antibodies were used for Western blot or FACS assays: mouse anti-CD36 (BD Biosciences, San Jose, CA), mouse anti-SRA-1 (R&D Systems), rabbit anti-SRB-1 (Thermoscientific), rat anti-LOX-1 and goat anti-CD206 (R&D Systems), rat anti-mouse ABCA-1 (Bio-Rad, Raleigh, NC), goat anti-ABCG-1and anti-Fox M1 (Santa Cruz, Dallas, TX), rabbit anti-PPAR, rabbit anti-ER, rabbit anti-ER (EMD Milipore, Temecula, CA) and mouse anti–actin and rabbit anti-iNOS (Cell Signaling Technology, Danvers, MA). Mouse FITC-conjugated anti-M mAb (clone M1/70), PE or FITC-conjugated F4/80 mAb, anti-L-PE were from eBioscience (San Diego, CA). anti-X-PE, anti-4-PE mAbs were from (BD Biosciences, San Jose, CA). The anti-D antibody was provided by Dr. Yakubenko and was previously described (27). Low-density lipoprotein/very-low density lipoprotein (LDL/VLDL) cholesterol was measured in mouse plasma using an HDL & LDL/VLDL Cholesterol Quantification Kit. Triglyceride levels were analyzed using Triglyceride Quantification Colorimetric/Fluorometric Kit (BioVision Research Products, Milpitas, CA). 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP), an ER antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-]pyrimidin-3-yl]phenol (PHTPP), a ER antagonist, were from Tocris Bioscience (Minneapolis, MN). Mouse IL-6, IL-12 p40/p70 and IL-10 Elisa kits were from RayBiotech (Norcross, GA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Modified LDL preparation LDL acetylation- LDL are combined with equal volume of saturated sodium acetate, mixed and cooled on ice. While constantly and slowly mixing the solution, the volume of the required acetic anhydride was added in 3 actions over 20 minute intervals. The modified LDL are then dialyzed overnight in the cold room against 100 fold excess of 09% NaCl, 0.05 EDTA, pH 7.4. The preparation is usually filtered through a 0.45 m filter. The protein content is determined by Lowry method and the extent of lysine modification is determined using TNBS assay (2,4,6-trinitrobenzene sulfonic acid). The protein content of the acetylated LDL.