Skip to main content
. 2020 Jun 1;27(11):3177–3195. doi: 10.1038/s41418-020-0571-7

Fig. 1. CHIP downregulates OTUD3 levels.

Fig. 1

a Livers from newborn CHIP WT and KO littermates (P0) and MEFs from CHIP WT and KO embryos (E13.5) were collected and the indicated proteins were detected by western blotting. b Representative images of OTUD3 immunohistochemical staining in liver tissues from newborn CHIP WT and KO littermates (P0). Scale bars: 50 μm. c Control or CHIP shRNAs (shCHIP #1, shCHIP #2) were packaged into lentiviral particles and transduced into H1299 cells. The positive clones were selected by puromycin (2 µg/ml). The indicated proteins were detected by western blotting. d H1299 cells stably expressing CHIP shRNA (shCHIP #1) were transfected with or without shRNA-resistant (sh-res) Myc-tagged CHIP plasmids for 48 h. The indicated proteins were detected by western blotting. e 293T cells were transfected for 48 h with Myc-tagged OTUD3 and increasing amounts of HA tagged CHIP plasmids. The indicated proteins were detected by western blotting. f 293T cells transfected with increasing amounts of HA tagged CHIP plasmids were collected 48 h post-transfection. The indicated proteins were detected by western blotting. g 293T cells were transfected for 40 h with Flag-tagged OTUD3 alone or together with Myc-tagged CHIP plasmids, and then treated with cycloheximide (CHX) (100 µg/ml) for indicated time period. The indicated proteins were detected by western blotting. The graph shows quantification of relative OTUD3 levels. h 293T cells were transfected for 40 h with or without HA tagged CHIP plasmids, and then treated with or without MG132 (20 µM) for 8 h. The indicated proteins were detected by western blotting. i 293T cells were transfected for 40 h with Flag-tagged OTUD3 alone or together with HA tagged WT CHIP, H260Q mutant CHIP or K30A mutant CHIP plasmids, and then treated with or without MG132 (20 µM) for 8 h. The indicated proteins were detected by western blotting. j 293T cells transfected with or without HA tagged WT CHIP, H260Q mutant CHIP or K30A mutant CHIP plasmids were collected 48 h post-transfection. The indicated proteins were detected by western blotting.