Fig. 3. AS160 controls the insulin-induced cell surface presentation of TβRI and TβRII.

(A) Increased phosphorylation of AS160 in response to insulin accompanies increased cell surface abundance of TβRI and TβRII. MEFs were treated with insulin and cell surface proteins were biotinylated. Neutravidin-affinity purified biotinylated proteins were immunoblotted for TβRI and TβRII. Phosphorylation of AS160 was assessed by immunoblotting for phosphoAS160-Thr⁶⁴². Whole cell lysates were immunoblotted for AS160, TβRI, TβRII, and GAPDH as loading control. Error bars indicate standard errors, based on three independent experiments. (B) siRNA-mediated decrease of AS160 in NMuMG cells results in increased TβRI and TβRII abundance at the cell surface, assessed by immunoblotting of affinity-purified biotinylated cell surface proteins. Whole cell lysates were immunoblotted for AS160, TβRI and TβRII, and GAPDH as loading control. Error bars indicate standard errors, based on three independent experiments. Pairwise comparison of relative abundance was performed using Wilcoxon rank-sum test. * p<0.05 (C) NMuMG cell lysates were subjected to sequential centrifugations to generate the LDM fraction of cell membranes, which was then fractionated by centrifugation in a 10–36% sucrose gradient. Twelve equal fractions spanning the gradient were analyzed by immunoblotting for AS160, TβRI, TβRII, EEA1, Rab4 and Rab11. Immunoblots are representative of three independent experiments. (D) Immunofluorescence of NMuMG cells expressing Flag-tagged AS160 and TβRI-EGFP, double stained with anti-Flag and anti-GFP. Scale bar, 10 µm. Images are representative of two independent experiments. (E) 293T cells expressing Flag-tagged AS160 and/or Myc-tagged TβRI were subjected to chemical crosslinking using DSP, and analyzed by immunoprecipitation and immunoblotting using the antibodies shown. Immunoprecipitation of TβRI resulted in co-immunprecipitation of AS160. Immunoblots are representative of two independent experiments. (F, G) AS160 silencing enhances autocrine TGF-β/Smad-induced transcription in NMuMG cells. In (F), cells were transfected with the Smad reporter plasmids 4xSBE or 3TP-Lux, and luciferase expression of cells, treated or not with SB431542, was normalized against the co-transfected Renilla-Lux reporter. In (G) Serpine1 mRNA (which encodes PAI-1) and Smad7 mRNA, expressed by cells treated or not with SB431542, were quantified by qRT-PCR and normalized against Rpl19 mRNA. Error bars indicate standard errors, based on three independent experiments. Statistical analysis was performed using Wilcoxon rank-sum test (* p<0.05, ** p<0.0083). In panels A, B, C and E: AP, affinity purification, IB, immunoblot, IP, immunoprecipitation. Ctrl, control.