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. 2017 Jul 6;8(46):80429–80442. doi: 10.18632/oncotarget.19035

Figure 3. GPC3 decreases IGF-1R ubiquitination.

Figure 3

(A) IGF-1R ubiquitination increased after GPC3 knockdown in HuH-7 cells. The cells were transfected with shGFP or shGPC3 and subsequently cultured in serum-free medium for 1 day. Immunoprecipitation was performed with anti-IGF-1Rβ, and western blot analysis was performed with anti-ubiquitin. Increased IGF-1R ubiquitination was observed in GPC3 knockdown cells (lane 2; p < 0.001, T test). Quantitation was also expressed as a fold-change relative to the control experiment. (B) IGF-1R ubiquitination decreased after GPC3 overexpression in PLC-PRF-5 cells. PLC-PRF-5 stable clones expressing either an empty vector or GPC3 were stimulated with IGF-1, immunoprecipitation was performed using anti-IGF-1Rβ, and western blot analysis was performed using anti-ubiquitin. IGF-1R ubiquitination levels were high in PLC-PRF-5 cells, but this increase was prevented through the overexpression of GPC3 (lane 3 and 4) with or without IGF-1 stimulation. (C) HEK293T cells were transfected with GPC3 or deleted GPC3 (ΔGPC3), together with ubiquitin, Grb10, and Nedd4. The cells were stimulated with IGF-1, and subsequently immunoprecipitation was performed using anti-IGF-1Rβ, and western blot analysis was performed using anti-ubiquitin. IGF-1 stimulation induced the ubiquitination of IGF-1R (lane 2), which was prevented by the overexpression of GPC3 (lane 4), but not ΔGPC3 (lane 6). The amount of IGF-1R was slightly decreased by ΔGPC3. All experiments were duplicated.