Gtx 129110

Cell lysate collected from nontransduced, transduced with Ad-Foxn1 or Ad-GFP keratinocytes were prepared in 200 μL RIPA buffer containing Protease Inhibitor cocktail (PhosStop, Roche; Protease Inhibitor, Sigma-Aldrich, St. Louis, MO, USA). Thirty milligrams of protein per sample separated on 12% SDS/PAGE gels and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA) were incubated with anti-BMP2 (1:1000, ab14933, Abcam, Cambridge, MA, USA), anti-IGF2 (1:800, GTX 129110, GeneTex, Irvine, CA, USA), anti-GAPDH (1:1500, ab 9484, Abcam, Cambridge, MA, USA) and anti-Actb (1:1000, ab 8226, Abcam, Cambridge, MA, USA) antibodies followed by fluorescent secondary antibodies (Alexa Fluor 680, 1:10,000, goat anti-mouse, A10038, Life Technologies, Thermo Fisher Scientific and Irdye 800, 1:10,000, 611-132-122, Rockland, Limerick, PA, USA). Bands were visualized using the ChemiDoc™ Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometric analysis was performed using Image Lab™ Software version 6.0 (Bio-Rad Laboratories, Inc.) (n = 3 samples per experimental group).

Cells were harvested in RIPA lysis buffer supplemented with a protease inhibitor cocktail, followed by immunoblotting with SOX2 antibody (1:1000 dilution, GTX101506, GeneTex), p-AKT (Ser473, 1:1000 dilution, GTX28932, GeneTex), AKT (1:1000 dilution, #1085-1, EPITOMICS), IGF2 (1:1000 dilution, GTX129110, GeneTex), IGF1R (1:1000 dilution, GTX50433, GeneTex), IGFBP1 (1:1000 dilution, GTX129006, GeneTex), and GAPDH (1:10,000 dilution, GTX100118, GeneTex).