Goat anti mouse or anti rabbit irdye conjugated antibodies

Tumors or cell lysates were harvested in RIPA lysis and extraction buffer (Thermo Fisher Scientific) with 1X HALT protease and phosphatase inhibitor cocktail (Thermo). Protein lysates were resolved on 4%–12% gradient SDS-PAGE gels and were transferred to nitrocellulose membranes, and incubated at 4°C overnight with the indicated antibodies diluted in 1:3 Odyssey Blocking Buffer in TBST (LI-COR). Antibodies were purchased from Cell Signaling Technology: phospho-ERK1/2 (RRID: AB_2315112), ERK1/2 (RRID: AB10695739), Cyclin D1 (RRID: AB_2259616), phospho-RB (S780, RRID: AB_330015; S807, RRID: AB_11178658), RB (RRID: AB_823629), phospho-MEK1/2 (RRID: AB_330810) MEK1/2 (RRID: AB_10695868), β-actin (RRID: AB_2242334), β-Tubulin (RRID: AB_823664). The anti-body for p16-INK4A (RRID: AB_2078303) was purchased from ProteinTech. Blots were incubated with secondary goat anti-mouse or anti-rabbit IRDye-conjugated antibodies (LI-COR Biosciences), and proteins were imaged with the Odyssey CLx imager (LI-COR Biosciences). Protein expression was quantified with the Odyssey Image studio software (Version 5.2.5), and normalized to the corresponding loading control.

Cells treated with the indicated doses of PF-573,228 or DMSO were harvested in CHAPS lysis buffer (10mM CHAPs, 50mM Tris (pH8.0), 150mM NaCl and 2mM EDTA) with 1× phosphatase and protease inhibitor cocktail (Roche). Protein lysates resolved on SDS-PAGE gels were transferred to Immobilon-FL membranes (Millipore) and incubated at 4°C overnight with the following antibodies: FAK, p130Cas (BD Bioscience); pY397FAK (Abcam); pY925FAK, PYK2, pY416Src, pY527Src Src, pY410p130Cas, ppERK 1/2, ERK 1/2, pS473Akt, pT308Akt, Akt, ppT180/Y182P38, P38 (Cell Signaling); pY861FAK, pS910FAK, pY402PYK2 (invitrogen); α-tubulin (CALBIOCHEM) diluted in 1:3 Odyssey® Blocking Buffer in TBST (LICOR). Blots were incubated with secondary goat anti-mouse or anti-rabbit IRDye-conjugated antibodies (LICOR), and proteins were imaged with the Odyssey CLx imager (LICOR).