Figure S3 Validation of mouse anti-Rab1 and rabbit anti Rab1 antibodies by Western Blot and immunofluorescence assays. from Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster

(a) Mouse anti-Rab1 S12085a was used in Western blotting to detect RFP-Rab1 and endogenous Rab1 proteins from either RFP-Rab1 males or wild type control Oregon-R males. (b) Western blot analyses of Rab1 depletion levels after RNAi knockdown using two different Rab1 antibodies respectively mouse anti-Rab1 S12085a and rabbit anti-Rab1 L12085a/169. Tubulin was used as loading control. (c) Quantification of band intensities in the Western blots using ImageJ software (version 1.42q; National Institute of Health, USA). The intensity of each band relative to the intensity of loading control (Tubulin), was normalized to the wild type control. The results are from three independent experiments. (d) Mouse anti-Rab1 S12085a was used to immunostain interphase-primary spermatocytes from wild type Oregon-R males and omt/Df mutant males. DNA was stained with DAPI. Note the absence of Rab1 signals in omt/Df. (e) Rab1 localization in DMel cells during cytokinesis. Cells were treated with Rab1 dsRNA (Rab1RNAi) or with Kanamycin dsRNA (control) for 72 hours and then fixed and stained with rabbit anti-Rab1 L12085a/169 to reveal endogenous Rab1, tubulin and DNA. Scale bars, 10 µm.



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