The degrees of 20-hydroxyecdysone (20E) were measured in the salivary glands of early wandering third instar larvae of control larvae (driver (gene

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The degrees of 20-hydroxyecdysone (20E) were measured in the salivary glands of early wandering third instar larvae of control larvae (driver (gene. nucleus and PBDB-T in the cytoplasm. High levels of rxYFP-CncC resulted in an aberrant morphology of salivary gland cells. (B). Visualization of dKeap1 and CncC in the prothoracic gland and in imaginal disc cells. The proteins indicated in the images were visualized in the tissues PBDB-T indicated above the images. Ectopically expressed rxYFP-dKeap1 was visualized by imaging intrinsic fluorescence in freshly dissected tissue (yellow, middle panels). rxYFP-dKeap1 was expressed under the control of the driver. Ectopic rxYFP-CncC was visualized by imaging intrinsic fluorescence in freshly dissected tissue (lower left) and endogenous CncC was visualized by immunostaining using anti-CncC antibodies (lower right). rxYFP-CncC was expressed under the control of the driver. Hoechst staining of the nuclei (blue) is Rabbit polyclonal to AP3 usually shown separately in the monochrome images to the right of each color image. Results: Both endogenous dKeap1 (Physique 1C) and the dKeap1 fusion were present within the nuclei of polyploid prothoracic gland cells and of diploid imaginal disc cells. Likewise, both the CncC fusion and endogenous CncC were predominantly nuclear in prothoracic gland cells and in imaginal disc cells. Thus, CncC and dKeap1 were localized to the nuclei in many different tissues and cell types. (C). Specificity of dKeap1 immunoreactivity. The midgut of control (larvae, and the band corresponding to endogenous dKeap1 was not detected by immunoblotting of extracts from these larvae, demonstrating the PBDB-T specificity of the anti-dKeap1 antibodies. (D). Specificity of CncC immunoreactivity. Extracts of early 1st instar larvae of the genotypes indicated above the lanes were analyzed by immunoblotting using anti-CncC and anti-tubulin antibodies as indicate below the blots. The bands corresponding to endogenous CncC are indicated. Results: The band corresponding to endogenous CncC was not detected by immunoblotting of extracts from larvae, demonstrating the specificity of the anti-CncC antibodies.(EPS) pgen.1003263.s001.eps (7.2M) GUID:?2CCDDC08-DB2F-424E-8065-00872B52BEAA Physique S2: Effects of CncC depletion on puff gene transcription and on larval ecdysteroid levels. (A). Effects of CncC depletion in larvae produced by two different cncC-RNAi sub-lines on transcription of ecdysone-regulated genes in salivary glands. The levels of the transcripts indicated below the bars were measured in salivary glands that expressed the shRNA targeting CncC under the control of the driver. The transcript levels were measured in larvae produced by two sub-lines that had been propagated separately for more than two years (and transgene, but lacked a GAL4 driver (open bars). To facilitate comparison of the transcript levels, the level of each transcript was normalized by the level of the transcript in the control larvae (or transcript. The data represent the means and the standard deviations from two individual experiments (*, p 0.05). (B). Effects of CncC depletion in the salivary glands on the level of 20E in the larvae. The levels of 20-hydroxyecdysone (20E) were measured in the salivary glands of early wandering third instar larvae of control larvae (driver (gene. (A). Effects of CncC depletion around the transcription of ecdysone biosynthetic genes in prothoracic glands. The levels of the transcripts indicated above the upper graphs were measured in the brain complexes from control larvae (driver (and sub-lines, demonstrating the genetic stability and reproducibility of these effects. (B). Effects of CncC depletion in the PG on Sad protein expression. The dissected brain complexes of control larvae (driver reduced Sad immunoreactivity in the PG. The polyploid nuclei of the PG were identified based on their PBDB-T large size compared to the diploid nuclei of the brain (right panels). The size and the number of nuclei in the PG were not altered by expression of the shRNA targeting CncC, suggesting that CncC depletion did not disrupt the overall structure of the PG (see also Physique 3C). (C). Effects.