Immunodetection of tubulin

Equal amounts of total protein were resolved on SDS–polyacrylamide gels and transferred to a PVDF membrane (0.2 μm pore, Bio-Rad) using a semi-dry electrotransfer (Bio-Rad). Gemin5 was immunodetected using anti-Gemin5 (Novus) antibody or anti-Xpress (Thermo Fisher Scientific). Commercial antibodies were used to detect RPL3 and RACK1 (Santa Cruz). P1 and P2 ribosomal proteins were detected with the monoclonal antibody 3BH5 [63 (link)]. Anti-H3A (abcam) and Histone H2A (Cell Signaling) were a kind gift from Dr. C. Gutierrez and Dr. E. Lecona. Immunodetection of tubulin (Merck) was used as loading control. The appropriate secondary HRP-conjugated antibodies (Thermo Fisher Scientific) were used according to the instructions of the manufacturer.

The protein concentration in the lysate was determined by the Bradford assay. Equal amounts of protein were loaded in SDS‒PAGE and processed for western blotting to determine the expression of the polypeptides using anti-Xpress antibody (Thermo Fisher Scientific, catalog R910-25, monoclonal, lot 2190234). The Clone name for Invitrogen anti-Xpress is reference 46-0528 (lot 2190234). RRID of mouse monoclonal IgG1 is AB_2556552. Immunodetection of tubulin (Merck) was used as a loading control. The anti-tubulin antibody Sigma is monoclonal DM1A (ascites fluid) (catalog T9026, lot 096k4777). Goat anti-mouse (H + L) secondary antibody from Invitrogen (Thermo Fisher Scientific, catalog 32430, lot VJ313743) was used according to the manufacturer’s instructions. The signal detected was performed in the linear range of the antibodies. The dilutions for anti-Xpress and anti-tubulin are 1:2000 and 1:4000, respectively. The dilution for the secondary antibody is 1:2000.