Fig. 4
FBXL6 stabilizes HSP90AA1 protein by promoting its K63-ubiquitination. a The protein and mRNA levels of HSP90AA1 from SMMC-7721 cells transfected with Flag-Con (EV) or Flag-FBXL6 indicated plasmids were detected by immunoblotting and real-time quantitative PCR, respectively. b The protein and mRNA levels of HSP90AA1 from Hep3B cells transfected with indicated plasmids were detected by immunoblotting and real-time quantitative PCR, respectively. c Western blot analysis of the WCL derived from SMMC-7721 cells or Hep3B cells infected with the indicated shRNA lentiviruses. d The protein and mRNA levels of HSP90AA1 from FBXL6+/+and FBXL6−/− SMMC-7721 cells were detected by immunoblotting and real-time quantitative PCR, respectively. e Western blot analysis of the WCL derived from SMMC-7721 cells transfected with EV or Flag-FBXL6ΔF-box plasmids. f FBXL6+/+and FBXL6−/− SMMC-7721 cells were treated with 20 μM cycloheximide (CHX) for the indicated time. The whole cell lysate was immunoblotted with anti-HSP90AA1 antibody. The quantification plot was based on scanning densitometry analysis using the Image J software. Relative protein levels were normalized to FBXL6−/− control group. g Western blot analysis of the WCL derived from FBXL6+/+and FBXL6−/− SMMC-7721 cells treated with 10 μM MG132 for 6 h. h SMMC-7721 cells transfected with EV or Flag-FBXL6 plasmids were harvested and lysed. WCL were immunoprecipitated by Tandem Ubiquitin Binding Entity 2 (TUBE2) resin for ubiquitinated proteins enrichment and immunoblotted as indicated. i SMMC-7721 cells were co-transfected Flag-FBXL6 and His-K63-ub for 36 h, cell lysate was subjected to immunoprecipitation by Ni+ purification. Immunoprecipitate was detected by immunoblotting using the indicated antibodies. j IHC analysis was performed on 87 specimens from HCC patients using anti-FBXL6 and anti-HSP90AA1 antibodies. Representative images of IHC staining of tumors of two HCC patients are presented. k The correlation study of FBXL6 and HSP90AA1 protein expression in 87 HCC samples is shown