Fig. 7

Competitive binding of GANC and HA to PSMD1 or PSMD2. a HEK293T cells were transfected with GANC plasmids tagged with Flag. 48 h later, the WCLs were incubated with anti-Flag magnetic beads and subjected to Western blotting to detect PSMD1 or PSMD2. b Purification and Coomassie staining analysis of GANC proteins. c Detection of pulldown proteins by Western blotting after incubating anti-Flag magnetic beads with GANC proteins, followed by incubation with PSMD1 or PSMD2 tagged with HA. d Schematic diagram of various truncations of PSMD1. e, f Western blotting for the detection of PSMD1 mutants in HEK293T cells transfected with PSMD1 truncations tagged with HA, along with HA plasmids tagged with Flag (e), or GANC plasmids tagged with Flag (f). WCLs were incubated with anti-Flag magnetic beads. g Schematic diagram of various truncations of PSMD2. h, i Western blotting for the detection of HA (h) or GANC (i) in HEK293T cells transfected with PSMD2 truncations tagged with HA, along with HA plasmids tagged with Flag, or GANC plasmids tagged with Flag. The red pentagons indicate the locations of various truncations of PSMD2. WCLs were incubated with anti-HA magnetic beads. j, k HEK293T cells transfected with HA plasmids tagged with Myc, followed by transfection with increasing amounts of GANC plasmids tagged with Flag, along with PSMD1 (j) or PSMD2 (k) plasmids tagged with HA. Cells were treated with MG132 (20 μM) for 12 h before collection. Western blotting was performed after incubating the WCLs with anti-HA magnetic beads. l, m Detection of pulldown proteins by Western blotting after incubating anti-HA magnetic beads with PSMD1 (l) or PSMD2 (m) proteins tagged with HA for 12 h, followed by incubation with HA protein tagged with His, and GANC protein tagged with Flag. Quantification was shown as mean ± SEM. n = 3 independent experiments. The Student’s t-test (unpaired, two-tailed) was used to compare two independent groups. **P < 0.01, ***P < 0.001, ****P < 0.0001. WCL whole-cell lysate