Ab133776

Cellular proteins were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined by the BCA method. A total of 40 μg of protein for each group was separated on 12% SDS-PAGE gels and transferred to 0.45-μm PVDF membranes (EMD Millipore). Membranes were then blocked with 5% BSA and incubated with primary antibodies at 4°C overnight against HPS70 (4876, CST), CD9 (ab92726, CST), CD63 (ab193349, Abcam), RBP4 (ab233138, Abcam), RPSA (ab133645, Abcam), RPS3(9538, CST), RPS20 (ab133776, Abcam), RPS14 (ab246916, Abcam), RPL4 (ab234829, Abcam), RPL13 (ab134961, Abcam), HSPD1 (ab46798, Abcam), HSPA8 (8444, CST), P-gp (13342, CST), p-cofilin-1 (3313, CST), cofilin-1 (5175, CST), PP1 (sc-7482, Santa Cruz), PP2A (9780, CST). Anti-β-actin (ab179467, Abcam) and anti-GAPDH (2118, CST) were used as the internal control. Anti-COX IV (11967, CST) was used as loading control for mitochondrial proteins. The membranes were then incubated at 37°C for 1 h with an HRP-conjugated secondary antibody (ab97051, Abcam). Bands were visualized by chemiluminescence according to the manufacturer’s protocols.

Whole-cell lysates were prepared by scraping cells in SDS sample buffer (125 mM Tris [pH 6.8], 20% glycerol, 4% SDS). Lysates were sheared with a 25G needle and boiled for 10 min at 95 °C. Protein concentration was determined by standard BCA protein assay (Pierce). Equal amounts of protein were separated on precast polyacrylamide gels (Invitrogen). Immunoblotting was done according to standard methods using IRDye800CW- and IRDye680-labeled secondary antibodies for detection on the Odyssey Infrared imager (LI-COR) or using horseradish peroxidase–conjugated secondary antibodies for detection by enhanced chemiluminescence (Supersignal and Supersignal West Pico Plus; Thermo Scientific). Primary antibodies used were against UBE2D (A615, Boston Biochem, 1:2000 dilution or 4330S, CST, 1:500 dilution), V5 (R960-25, Invitrogen, 1:1000 dilution), HA (MMS-101R, Covance, 1:1000 dilution), FLAG (F3165, Sigma, 1:1000 dilution), RPS20 (ab133776, Abcam, 1:1000 dilution), Histone H3 (ab1791, Abcam, 1:10,000 dilution), CRABP1 (HPA017203, Sigma, 1:1000 dilution), TSPAN8 (A06997-1, SanBio, 1:1000 dilution), GAPDH (PA1-987, ThermoFisher, 1:5000 dilution), HSP90 α/β (sc-7947, Santa Cruz, 1:1000 dilution), β-actin (A5316, Sigma, 1:10,000 dilution), and γ-tubulin (T6557, Sigma, 1:10,000 dilution).