The SNX-BAR proteins are needed to prevent lysosomal degradation of the activated IGF1R. (A) HeLa cells, clonal SNX1/2 and SNX5/6 double-KO cells, and two VPS35-KO cell lines were serum starved and treated with 10 nM IGF-1 for the indicated periods. The level of endogenous IGF1R and the INSR were detected by Western blotting, and the IGF1R degradation kinetics were quantified over four independent experiments. (B) HeLa cells, clonal SNX1/2, and SNX5/6 double-KO cells as well as VPS35-KO cell lines were transduced with a lentiviruses expressing human IGF1R-myc, serum starved, and then treated with 10 nM IGF-1 for 1 h. The IGF1R and endogenous LAMP1 were stained by immunofluorescence, and the colocalization was quantified across 16 images acquired in two independent experiments. (C) HeLa cells and SNX1/2 and SNX5/6 double-KO cell lines were serum starved and treated with 5 µg/ml insulin for the indicated periods, and the level of the endogenous INSR was quantified by Western blotting over three independent experiments. (D) SNX5/6 double-KO cells were transduced with lentiviruses expressing GFP-SNX5 or GFP-SNX6 and with GFP only as a control. The cells were serum starved and treated with 10 nM IGF-1 for the indicated periods, followed by Western blot–based detection of the levels of the endogenous IGF1R. The level of the endogenous IGF1R in starved cells (top bar graph) as well as the degradation kinetics in IGF-1–treated cells were quantified over four independent experiments. (E) HeLa cells and clonal SNX5/6 double-KO cells were serum starved and treated with 10 nM IGF-1 for the indicated periods. One set of the SNX5/6 KO cells was treated with the lysosomal inhibitor bafilomycin-A1. The degradation kinetics were quantified over four independent experiments. Bars, 10 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.