Fig. 3: USP32 interacts with BAG3 and upregulates the protein level of BAG3.

A The number of differentially expressed genes was screened by iTRAQ-based proteomics after overexpression of USP32 in H1299 cells. B H1299 cells were transiently transfected with Myc vector or Myc-USP32 expression plasmid for 48 h. Proteins were extracted from the cells and separated on a gel, which was then stained with Coomassie brilliant blue R-250. A framed protein band was evaluated by mass spectrometry analysis. C Representative mass spectral peaks of BAG3 interacting with Myc-USP32. D Docking model of USP32 and BAG3 proteins and surface map of their interfacial residues (USP32, blue; BAG3, yellow; hydrogen-bonding interactions, dotted line). E Co-localization studies of three cells of NSCLC using anti-USP32 antibody (1:100, green) and anti-BAG3 antibody (1:100, red) followed by DAPI nuclear counterstaining (blue). Merged images of USP32 (green) and BAG3 (red) with DAPI (blue) are also shown. Scale bar:50 μm. F, G Exogenous interaction of USP32 with BAG3. HEK293T cells were cotransfected with Myc-USP32 and/or Flag-BAG3 plasmids. Cell lysates were immunoprecipitated with the indicated antibodies and then immunoblotted with anti-Flag antibody (F) or anti-Myc antibody (G). H, I Endogenous interaction of USP32 with BAG3. A549 and H1299 cell lysates were immunoprecipitated with anti-USP32/BAG3 antibody or IgG antibody, followed by immunoblotting with anti-BAG3/USP32 antibody. J, K USP32 and BAG3 protein levels were detected by immunoblotting in USP32 overexpressed and knockdown overexpressed cells. **p < 0.01; ***p < 0.001.