Figure 5
From: Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis
Interaction of Gpnmb with calnexin in hepatic macrophages and stellate cells of the liver.
(a) Western blot analyses of LI90 cells transfected with p3xFLAG-mGpnmb or p3xFLAG-Vector by using anti-Gpnmb antibody. (b) Soluble proteins with FLAG tag were isolated and analyzed by SDS-PAGE and silver stain. Visible bands (black arrow) were excised and analyzed with LC-MS/MS and calnexin was identified. (c) Western blot analyses re-probed with anti-calnexin antibody using the membranes in panel (a,d) Calnexin was co-immunoprecpitated with FLAG-tagged Gpnmb protein. (e) Double immunofluorescence staining of calnexin and Gpnmb. (f) We used Duolink proximity ligation assay to identify the interaction between Gpnmb and Calnexin. The red signals showing the interaction between Gpnmb and Calnexin were mainly detected in F4/80-positive cells. Scale bar: 50 μm. (g) Cell lysates derived from stromal vascular fractions were immunoprecipitated with anti-calnexin antibody and immunoblotted with anti-Gpnmb antibody.
