Protein lysates from frozen tissues were prepared and Western Blots performed as previously described [41 (link)] using 10 to 25 µg total protein depending on the blot. Antibodies used include the following from Cell Signaling Technologies (Danvers, MA, USA; all of them used at 1:1000): anti-pAKT S473 (9271), anti-AKT (4685), anti-p4EBP1 (9459), anti-4EBP1 (9452), anti-pMAPK (9101), anti-MAPK (9102), anti-PTEN (9559), anti-FAK (3285), anti-VEGFR2 (2479) and anti-PDGFR-β (3169). Other antibodies were also used at 1:1000 except where indicated: anti-PTPN12 (Abcam, Cambridge, MA, USA; ab76492), anti-VEGFR1 (Abcam; ab32152), anti-VEGFR3 (Thermo Fisher Scientific; PA5-16871), anti-CD31 (Abcam; ab28364), anti-actin (Sigma-Aldrich; A5441, 1:10000), anti-VE-cadherin (Santa Cruz, Dallas, TX, USA; sc-6458), anti-pTyr 4G10 (Millipore, Billerica, MA, USA; 05-321), anti-pFAK (Sigma; F7926). Blots were imaged on a BioSpectrum Imaging System (UVP, Upland, CA, USA) and quantification of bands was performed using the instrument software.
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