Anti vegfr1

Western blot analyses were performed as previously described (X. P. Chen et al., 2016) with the following primary antibodies: anti‐endocan (0.1 μg/ml; R&D Systems); anti‐ERK1/2 (1:1,000; Cell Signaling Technology); anti‐p‐ERK1/2 (1:1,000; Cell Signaling Technology); anti‐VEGF (1:1,000; Abcam); anti‐VEGFR1 (1:1,000; Abcam); anti‐VEGFR2 (1:1,000; Cell Signaling Technology); and GAPDH (1:1,000; Cell Signaling Technology).

Mouse brain tissues were homogenized in ice-cold RIPA lysis buffer (1 × PBS buffer containing 1% NP-40, 0.5% Na deoxycholate, and 0.1% SDS) with a protease & phosphatase inhibitor cocktail (Thermo Fisher, cat#: UG280144) [47 (link)]. Protein concentrations were determined using the BCA protein detection kit (Thermo Fisher, cat#: 23227), and equal amounts of protein (10 µg per lane for tissue lysates) were resolved on denaturing 10% SDS–PAGE gels and transferred by electroblotting to PVDF membranes (Millipore, cat#: IPVH00010, Burlington, MA). Membranes were incubated with anti-VEGFR1 (Abcam, cat#: ab2350, 1:500), anti-VEGFR2 (Abcam, cat#: ab221679, 1:1000), anti-Cgn (Sigma, cat#: HPA027657, 1:500, St. Louis, MO), anti-ZO-1 (Invitrogen, cat#: 61–7300, 1:1000, Carlsbad, CA), anti-Claudin 5 (Invitrogen, cat#: 34–1600, 1:1000), or anti-Actin (Millipore, cat#: MAB1501R, 1:5000). The membranes were washed with PBST (0.2% Tween-20 in PBS), incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Earthox, cat#: E030110-01, 1:20,000, Millbrae, CA) or HRP goat anti-rabbit IgG (Earthox, cat#: E030120-01, 1:20,000) for 1 h, washed again, and incubated with ECL detection reagents (Millipore, cat#: WBKLS0500). Densitometry analysis was performed using the ImageJ (version 1.52p, NIH) software.

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.