Ab227456

To detect the CEP70 protein, testes of Cep70^−/− and WT male mice were pulverized in liquid nitrogen and collected in 500 μL ice-cold Radio Immunoprecipitation Assay Lysis Buffer (RIPA) buffer containing protease inhibitors. After that, the lysates were diluted with 2× Laemmli sample buffer (Bio-Rad, 1610737), and boiled in water for 10 min. The protein samples were separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE, 10% acrylamide running gel) and then electrically transferred to polyvinylidene fluoride (PVDF) membranes. After transfer, the PVDF membranes were blocked with 5% skimmed milk in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then immunoblotted at 4 °C overnight with Anti-CEP70 antibody (Abcam, ab227456, 1:1000, Rabbit) and Anti-β-actin mouse monoclonal antibody (TransGen Biotech, HC201, 1:1000). After washing in TBST, the membranes were incubated for 1 h with Anti-rabbit or mouse HRP-conjugated secondary antibody (1:3000). Finally, protein bands were visualized by an enhanced chemiluminescence detection system (Tanon-5200). Western blot images were processed using ImageJ software (Wayne Rasband, USA).

The following antibodies were used for localization studies and Western blot: ALMS1 (1:1500–2000, rb, ab84892 Abcam; 1:500, rb, NB100-97823, Novusbio), ARL13B (1:200, ms, 73-287, Neuromab; rb, 17711-1-AP, Proteintech); TUBGCP2 (1:100, rb, 25856-1-AP, Proteintech), CEP70 (1:500, rb, ab227456, Abcam), acetylated tubulin (1:250, ms, ab24610, Abcam), CEP250 (1:200, rb, 14498-1-AP, Proteintech), Centrin-3 (1:500, rb, PA5-35865, Thermo), Centriolin (1:200, ms, sc-365521, Santa Cruz), RPGR (1:500, rb, HPA001593, Sigma Aldrich). Secondary Antibodies were Alexa488 goat anti-mouse (A11029, Molecular probes) and anti-rabbit (A11034, Molecular Probes), Alexa568 goat anti-mouse (A11031, Molecular probes) and anti-rabbit (A11036, Molecular Probes). For Western blot FLAGM2-HRP (Sigma) was used.