Figure 1.

Characterization of R‐cells (igf1r−/−) stably expressing wild type (WT) or triple SUMO‐sites mutated (TSM) IGF1R. (A) Relative IFG1R mRNA transcription levels in selected transfected R‐cell clones were determined by qRT‐PCR. Two cell lines with equal expression of WT (WT‐2C4) and TSM (TSM‐2D4) IFG1R mRNA (marked with arrows) were chosen for further investigation and named R‐WT and R‐TSM, respectively. R‐puro was an empty vector control. (B) IGF‐1R and pre‐IGF1R protein expression in R‐puro, R‐WT, R‐TSM cells compared to the cancer cell lines MCF7 (breast cancer), H1299 (lung cancer), HCT116 (colon cancer) were determined by immunoblotting (IB) with anti‐IGF‐1Rβ. GAPDH was blotted as loading control. (C) SUMOylation of IGF‐1R in R‐puro, R‐WT, and R‐TSM cell lines were determined by immunoprecipitation (IP) of IGF‐1R and IB for SUMO1. The three predicted SUMO‐IGF‐1R bands are indicated by arrows. Re‐blot of IGF‐1Rβ as an input IP control. (D) IGF‐1R tyrosine kinase activity was assessed by receptor and substrate phosphorylation. IGF‐1R was IPed from lysates from R‐puro, R‐WT, and R‐TSM cells that had been subjected to 36‐h serum starvation with or without subsequent 10‐min IGF‐1 stimulation, and blotted with anti‐phospho‐tyrosine antibody. Re‐blot of IGF‐1R β served as input control. The lysates were also directly blotted for phosphorylated Erk (pErk) and phosphorylated Akt (pAkt). GAPDH was used as loading control. (E) R‐puro, R‐WT, and R‐TSM cells were fractionized and the nucleus portions were immunoblotted with anti‐IGF1Rβ. Histone 3 was blotted as loading control. (F) Association of IGF‐1R and InsR was investigated by co‐IP. IGF‐1R IPs from R‐puro, R‐WT, and R‐TSM cell lysates were blotted for InsRβ. Re‐blot of IGF‐1Rβ served as control of IP. (G) Co‐localizations between IGF‐1R and InsR were visualized by PLA (red dots) in R‐puro, R‐WT, and R‐TSM cells. IGF‐1R and cell nuclei were counterstained with Alexa Fluor® 488 (Green) and DAPI (Blue), respectively. Data are representative of 3–5 experiments