Western lightning chemiluminescent kit

Equal amounts of proteins were resolved in either 8 or 10% SDS-PAGE and electroblotted onto polyvinylidine difluoride membrane (PVDF) using semi-dry blotting system (Trans-Blot Transfer medium; Bio-Rad Laboratories). Membranes were blocked in 5% BSA in PBS containing 0.1% Tween-20 and incubated overnight at 4°C with antibodies against rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000), rabbit anti-ERK1/2 (1:1000), rabbit anti-phospho-AKT (Ser473) (1:500), rabbit anti-AKT (1:1000) from Cell Signaling Technology (Danvers, MA); mouse anti-EGFR (1:200) and anti-pan actin (1:50 000) antibodies from Neomarker (Fremont, CA). After several washes, membrane was incubated with either goat anti-rabbit or goat anti-mouse horseradish peroxidase conjugated secondary antibodies (DakoCytomation, Denmark) (1:10 000). Specific protein bands were visualized with an enhanced chemiluminescence using Western Lightning chemiluminescent kit (Perkin-Elmer, MA).

Equal amounts of protein lysates from MSCs were resolved by either 6% or 15% SDS/PAGE and electroblotted onto polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Nonspecific binding sites were blocked for 1 h with PBS containing 5% BSA and 0.1% Tween‐20 at room temperature (RT). The membrane was then immunoblotted against anti‐IGFBP2 clone C‐18 (dilution 1 : 1000; Santa Cruz Biotechnology Inc.), IGF‐2 (dilution 1 : 500; R&D Systems), IGF‐1R and IGF‐2R (dilution 1 : 200; R&D Systems) at 4 °C. Following washing and incubation with either rabbit anti‐goat or goat anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (dilution of either 1 : 5000 or 1 : 10 000; DakoCytomation, Denmark), proteins of interest were visualized with an enhanced chemiluminescence using Western Lightning chemiluminescent kit (Perkin‐Elmer, MA, USA). Normalization was performed using antibody against actin (dilution 1 : 20 000; Thermo Fisher Scientific, Fremont, CA) or tubulin (dilution 1 : 5000; Santa Cruz Biotechnology Inc.). Semiquantitation analysis, to normalize the protein bands against respective loading controls, was performed using Metavue software v6.1 (Molecular Devices, LLC, CA).