Hrp mouse igg kappa binding protein

Cells were collected and lysed in 2× RIPA buffer+2× Protease Inhibitors (Roche, #4693159001). The protein concentration was determined with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, #23225) according to the manufacturer's protocol. Equal amounts of lysates were loaded on a 14% or 18% SDS-Page for size fractionation. After electrophoresis, proteins were immunoblotted on a PVDF-membrane (Sigma-Aldrich, #3010040001) for 1 h at 100 V and blocked with 5% milk in Tris-buffered saline and 1% Tween20. Proteins were detected with rabbit anti-ARPC5 (Novus, #NBP2-67350, lot #HL0702, 1:1000), rabbit anti-ARPC5L (Abcam, #ab169763, lot #GR121833-4, 1:2000) or mouse anti-ARPC3 (anti-Arp2/3 Complex Antibody Clone 13c9, Millipore, #MILL-MABT95, lot3283230, 1:5000). HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, #7074S, 1:3000) and HRP mouse IgG kappa binding protein (Santa Cruz, sc-516102, 1:3000) were used as secondary antibodies before detection with ECL chemiluminescent substrate (Cell Signaling Technology, #12630S, 1:1). Anti-Tubulin (Proteintech, #HRP-66031, 1:5000) was used as a loading control and detected as a 55 kDa band.

Anti-RBX1 antibody (ab133565, 1:1000 dilution for immunoblotting and 1:100 for IHC) was purchased from Abcam. Anti-POLR2A antibody (sc-47701,1:5000 dilution for immunoblotting and 1:200 for IHC), anti-CUL1 antibody (sc-17775, 1:1000 dilution), anti-CUL2 antibody (sc-166506, 1:1000 dilution), anti-CUL4A/4B antibody (sc-377188, 1:1000 dilution), anti-CUL5 antibody (sc-373822, 1:1000 dilution) and anti-CUL7 (sc-53810, 1:1000 dilution) antibody were obtained from Santa Cruz. Anti-Ki67 antibody (#12075, 1:100 dilution), anti-cleaved Caspase-3 (Asp175) (#9664, 1:1000 dilution), Anti-SKP2 antibody (#2652, 1:1000 dilution), Anti-VHL antibody (#68547, 1:1000 dilution) and Anti-CUL3 (#2759, 1:1000 dilution) were purchased from Cell Signaling Technology. Anti-p53 (sc-126, 1:1000 dilution), anti-actin (sc-1616, 1:5000 dilution), HRP-anti-mouse IgG (sc-2055, 1:5000 dilution), HRP-mouse IgG kappa binding protein (sc-516102, 1:5000 dilution) and HRP-anti-rabbit IgG (sc-2054, 1:5000 dilution) were purchased from Santa Cruz.