Figure 3.
Colocalization of CBX8 and BMI1 in Polycomb bodies. (A) TIG3-T cells were infected with control (pRS) or shRNA targeting CBX8 (pRS-CBX8). Puromycin-selected cells were fractionated into soluble and insoluble proteins. Input (total lysate), insoluble and soluble protein fractions were loaded as indicated, and the levels of CBX8, histone H3, GAPDH and BMI1 were revealed by Western blot analysis. (B) Immunodepletion of BMI1 and CBX8 from TIG3-T protein extracts. Three successive immunoprecipitations (IPs) were performed (IPs 1–3), followed by IP of CBX8 in the BMI1-depleted extract and of BMI1 in the CBX8-depleted extract. Extracts before depletion (BD) and after depletion (AD, after the third IP) were loaded next to each other to reveal differences in protein levels (*background, crosslinked IgG heavy and light chains, 75 kDa). (C) Colocalization of CBX8 with either BMI1, RING1B, HPH1 or HPH2 in pre-extracted U2OS cells. U2OS cells grown on coverslips were pre-extracted, fixed and incubated with rabbit anti-CBX8 in combination with either mouse anti-BMI1, anti-RING1B, anti-HPH1 or anti-HPH2. Anti-rabbit Alexa-594 and anti-mouse Alexa-488 were used for detection. Coverslips were stained with DAPI, mounted and analyzed by confocal microscopy. (D) U2OS cells grown on coverslips were cotransfected with H2B-GFP and the indicated shRNA constructs. Cells were pre-extracted and fixed as in (C). For detection of BMI1, RING1B or HPH1 and CBX8, anti-rabbit Alexa-650 and anti-mouse Alexa-568 were used as secondary antibodies. After staining, coverslips were mounted and analyzed by confocal microscopy.