Anti phosphotyrosine py99

The following mouse monoclonal antibodies were used: anti-phosphotyrosine PY99 (Santa Cruz Biotechnology), anti-phosphotyrosine (4G10), anti-α9β1 antibody (Y9A2), anti-integrin α4 (HP2/1), anti-total β1 (MAB1959), anti-active β1 (12G10), anti-myc tag (4A6), anti-v-Src (OP07), anti-PTEN (6H2.1), anti-pan-actin (all from Millipore), activating β1-integrin antibody (TS2/16, Thermo Scientific), anti-Yes, anti-Fyn, anti-Hck, anti-Crk (all from BD Biosciences), anti-cellular/EDA fibronectin antibody (FN-3E2, Sigma). The following rabbit polyclonal antibodies were used: anti-cortactin (ab11066 from Abcam, for G361 lysates) and anti-cortactin phospho-Y470 (ab51703, from Abcam, or sc-101661 from Santa Cruz), anti-cortactin phospho-Y421 and anti-Src family kinases (SFK) phospho-Y416 (both from Cell Signaling), anti-arg, anti-Pyk2, anti-FAK (all from Millipore), anti-FAK phospho Y397 (Invitrogen), and anti-fibronectin (R2/7, made against bovine plasma fibronectin53 (link)). Secondary antibodies used were fluorochrome-conjugated antibodies (Alexa Fluor 488 and 546, Invitrogen) and horseradish peroxidase (HRP)-conjugated antibodies (Dako).

BLA and IL brain tissue samples were removed and lysed with TNE buffer. The protein concentration of each sample was detected by the BCA reagent (Thermo). For analysis of TrkB phosphorylation (p-TrkB), 5 mg protein was utilized for immunoprecipitation with the rabbit anti-TrkB antibody (1:200, Millipore), followed by immunoblotting with anti-phospho-tyrosine pY99 (1:3000, Santa Cruz), goat anti-TrkB (1:2000, R&D) and rabbit anti-β-actin (1:1000, Sigma) antibodies. Ratios of p-TrkB/total TrkB derived from control groups were normalized to 1.0.

Anti-PDGFRβ (958) and anti-phosphotyrosine (PY99) antibodies were purchased from Santa Cruz Biotechnology, Dallas, Texas, USA. The anti-PDGFRβ (AH 17.2) monoclonal antibody was produced as described [24 (link)]. Anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PLCγ1 (Tyr783) and anti-PLCγ antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA. Anti-calnexin antibody was purchased from Enzo® Life Sciences, Farmingdale, NY, USA. Secondary antibodies used in western blot experiments were purchased from Cell Signaling Technology. The secondary antibody coupled to phycoerythrin used in flow cytometry was purchased from Jackson ImmunoResearch, West Grove, PA, USA. Fluorescent secondary antibodies were purchased from LI-COR Biosciences, Lincoln, NE, USA. PDGF-BB was purchased from PeproTech, Rocky Hill, NJ, USA. Imatinib was purchased from LC Laboratories (Woburn, MA, USA). Cycloheximide was purchased from Sigma. ATP was purchased from Fermentas (Thermo Fisher Scientific, Waltham, MA, USA). EZ-Link Sulfo-NHS-Biotin and streptavidin agarose beads were purchased from Pierce (Thermo Scientific).