Fig. 4
From: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia
GRP75 binds to and stabilizes ANT2 by decreasing its ubiquitination level. a Identification of endogenous GRP75-interacting proteins. Extracts from the iWAT of YES2 tumour-bearing mice were incubated with protein A/G magnetic beads conjugated with an anti-GRP75 antibody. The eluted proteins were resolved by SDS‒PAGE and visualized by silver staining. The differential gel piece framed by the red rectangle was analysed by mass spectrometry. b Coimmunoprecipitation of endogenous ANT2 and GRP75 in the iWAT of YES2 tumour-bearing mice. Anti-IgG was used as a negative control. c Coimmunoprecipitation of GRP75 and ANT2 in differentiated 3T3-L1 adipocytes transfected with Myc-GRP75 plasmids. d PLA signals and quantification of the combination of anti-GRP75 and anti-ANT2 in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-overexpressing EVs. DAPI, nuclei. Scale bar, 10 μm. e Immunoblotting with anti-GST antibodies was used to detect the binding of recombinant His-ANT2 to GST-GRP75. f Coimmunoprecipitation of ANT2 and truncated domains of GRP75 (bottom panel). Flag-tagged wild-type GRP75 (GRP75-FL) or truncated GRP75 (GRP75-N and GRP75-C) were transfected into HEK293T cells. g Immunoblots (left panel) and real-time qPCR analysis (right panel) of ANT2 and GRP75 in differentiated 3T3-L1 adipocytes transfected with control vector (NC) or Myc-GRP75 plasmids. h Evaluation of the ANT2 half-life in GRP75-overexpressing adipocytes treated with cycloheximide. The indicated proteins were analysed by immunoblotting, and the quantification of ANT2 protein relative to the control β-actin by ImageJ software is shown at the bottom. i Ubiquitination assays of exogenous ANT2 in lysates from differentiated 3T3-L1 adipocytes transfected with NC or GRP75 plasmids. The data are presented as the mean ± SEM. The exact P values were tested with an unpaired two-tailed Student’s t test in (g)
