Anti igf1r

As previously described, to investigate the activity of the iNOS promoter, RAW 264.7 macrophages (10⁵ cells per well) were plated in 48-well polystyrene plates55 and transfected via Lipofectamine 2000 reagent (Invitrogen) by way of the manufacturer’s instructions. Cells were transfected with 1 μg pTK-3XNS or pTK-3XS (kindly provided by Dr. David Geller) plus plasmid pRL-CMV (Promega) that was the co-reporter for the normalization of experimental variations. Cells transfected with pTK-3XNS or pTK-3XS and pRL-CMV were treated with IGF-I (50 ng/ml; R&D Systems), IFN-γ (10 ng/ml; R&D Systems), or anti-IGF-1R (5 μg/ml; R&D Systems). Unstimulated cultures were included as controls. After these treatments, cells were washed with PBS, lysed according to the Dual Luciferase System Protocol (Promega), and analyzed in the TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). Luciferase activity was measured using the Luciferase Assay System (Promega), according to the manufacturer’s protocol. Each transfection was performed in triplicate, and three independent experiments were conducted.

Rabbit IgG (catalog AB-105-C), anti-IGF1 (catalog AF791), and anti-IGF1R (catalog MBA391-100) were purchased from R&D Systems. αMEM was from Thermo Fisher Scientific and FBS from Sigma-Aldrich. An anti–p-Akt antibody (catalog 9271) was purchased from Cell Signaling Technology. An anti-p16^INK4A antibody (catalog 03119) was purchased from GeneTex.