Igf2r

DMEM for cell culture was purchased from Corning (Corning, NY, USA). Fetal bovine serum, protease inhibitor cocktail, and αSMA antibody were from Sigma-Aldrich (St. Louis, MO, USA). IGF1R inhibitor I-OMe-Tyrphostin AG 538 was from Calbiochem (San Diego, CA, USA). Recombinant human IGF-II and anti-IGF-II antibody were purchased from R&D Systems (Minneapolis, MN, USA). Collagen, Fibronectin, and GAPDH antibodies were from Santa Cruz (Santa Cruz, CA, USA). IGF1R, IGF2R, IR-β, phospho-SMAD and total SMAD antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine^® 2000 was purchased from Invitrogen (Carlsbad, CA, USA). IGF1R, IR, and isotype control antibodies used in neutralization experiments were obtained from GroPep Bioreagents (Thebarton SA 5031, Australia). IGF1R, IGF1R, IR, and scrambled siRNA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HRP-conjugated anti-mouse IgG was from Promega (Madison, WI, USA) and anti-rabbit-HRP IgG was from GE Healthcare Life Sciences (Chicago, IL, USA).

Immunohistochemistry was performed as previously described [14 (link),15 (link)]. Briefly, after deparaffinization and rehydration, tissue sections (5 μm thick) were subjected to antigen retrieval, followed by peroxidase block and treatment with a blocking reagent for nonspecific binding. The slides were then incubated overnight with primary antibody at 4°C in a humidified chamber. The following primary antibodies were used: IGF-1 (Santa Cruz Biotechnology, Santa Cruz, CA, dilution 1:100), IGF-2 (Abcam, Cambridge, MA, 1:500), IGF-1R (Cell Signaling, Danvers, MA, 1:50), and IGF-2R (Cell Signaling, 1:50); followed by peroxidase-conjugated secondary antibody. Normal kidney served as positive controls for IGF-1, IGF-1R, and IGF-2. Negative controls included omission of primary antibody.
All immunohistochemistry evaluation was performed in a blinded manner by an attending pathologist. The slides were scored semiquantitatively. A score of 0 indicated no staining, 1 indicated focal weak staining, 2 indicated moderately positive staining, and 3 indicated intensely positive staining. Tumors with strong staining (scores 2 to 3) were grouped, and tumors with weak or no staining (scores 0 to 1) were grouped. All analyses were performed in triplicate.