P0013

For IB analysis, cell lysis buffer (P0013, Beyotime) supplemented with PMSF (ST506, Beyotime) and phosphatase inhibitor cocktail was used to lyse the cells (P1081, Beyotime). Proteins were isolated using polyacrylamide gel electrophoresis with sodium dodecyl sulfate and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk and the indicated primary antibodies at 4 °C overnight. After being washed three times with TBST buffer, the cells were incubated with the HRP-conjugated secondary antibodies (SA00001-1, SA00001-2; Proteintech) and detected by using chemiluminescence (34580, Thermo Fisher).
For IP analysis, cell lysis buffer (P0013, Beyotime) containing PMSF and phosphatase inhibitor cocktail was used to lyse the cells for IP analysis (P1081, Beyotime). Cell lysates were first precleared with protein A/G agarose, and then were incubated with the indicated primary antibodies at 4 °C overnight. The next day, 40 μl of protein A/G-agarose beads were added and incubated for 4 h at 4 °C. After washing three times, the mixture was resuspended. After boiling and centrifugation to pellet the agarose beads, supernatants were subjected to IB analysis.

For immunoblot analysis, cells were washed twice with ice-cold PBS, scraped, pelleted and lysed in the cell lysis buffer for Western and IP (P0013, Beyotime). After incubation for 10 min on ice, cell lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. Protein concentration of lysates was determined by BCA protein assay kit (P0010, Beyotime) and the lysates were adjusted with cell lysis buffer. Proteins were separated by 8–15% SDS-PAGE transferred to PVDF membrane (Millipore). The blots were blocked for 1 h at room temperature with 5% non-fat dry milk in TBST (TBS and 0.05% Tween-20). Incubation with specific primary antibodies was performed in blocking buffer overnight at 4 °C. Goat anti-mouse IgG or goat anti-rabbit IgG conjugated to HRP was used as secondary antibody. The blots were developed by Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) according to the manufacturer’s instructions. For immunoprecipitation, transfected cells were lysed in the Western and IP buffer (P0013, Beyotime). Cell lysates were incubated with Flag M2 beads (A2220, Sigma) or anti-Myc (9E10) beads (sc-40AC, Santa Cruz) and were analyzed by SDS-PAGE and blotted with indicated antibodies.

The cells were lysed using a non-denaturing lysis buffer (P0013, Biyuntian, Beijing, China). Immunoprecipitation (IP) was performed using protein A/G magnetic beads (Sigma, Shanghai, China). The IP-grade antibodies, including anti-JMJD1C (17-10262, Sigma, Shanghai) and anti-Ran (ab155103, Abcam, Shanghai, China) were used to enrich the proteins of interest. Rabbit IgG antibody was used as a control.