Fig. 3: USP35 interacts with and deubiquitinates BRPF1.

A Vector or FLAG-HA-USP35 plasmid was transfected in C4-2b cells for 24 h. After treatment of 10 μM MG132 for 4 h, cell lysate was immunoprecipitated with anti-FLAG and anti-HA beads, and the proteins interact with USP35 were screened. Silver staining picture was shown in the left panel and representative proteins were selected to show in the right panel. B The Co-immunoprecipitation (co-IP) assay was conducted the detect the endogenous interactions between USP35 and BRPF1 proteins in C4-2b. C Western blotting assay showed the decreased BRPF1 proteins in USP35-deleted C4-2b cells relative to parental control cells. The qPCR assay showed the mRNA levels of BRPF1 in three groups. D The C4-2b cells were transfected with increased amounts of Myc-USP35 plasmids and the protein levels of BRPF1 were detected by western blotting assay. The corresponding BRPF1 mRNA levels were also detected by RT-qPCR assays. E The parental and USP35-loss C4-2b cells were treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points. At each time point, the intensity of BRPF1 was normalized to the intensity of actin and then to the value at 0 h. F In contrast, the CHX assay revealed that USP35 overexpression could prolong the half-time of BRPF1 proteins in cells transfected with USP35 plasmids relative to control cells. G The in vivo ubiquitination assay was conducted in cells transfected with Flag-BRPF1, Myc-USP35 (WT, or CA), or HA-ub. The cells were treated with 20 μM MG132 for 8 h, and western blotting showed the products. H The C4-2b cells were co-transfected with Flag-BRPF1 and Myc-USP35 (WT, or CA), respectively. The western blotting and RT-qPCR assays were used to detect the protein and mRNA levels of BRPF1 in the indicated groups. * p < 0.05, ** p < 0.01, *** p < 0.001.