Anti plcγ

Commercial antibodies included: anti-TrkB (BD Biosciences, New Jersey, NJ, USA; #cat 610102), anti-TrkB (Millipore, Burlington, MA, USA; #cat 07-225), anti-phospho-TrkB (Tyr 515) (Sigma, St Louis, MO, USA; #cat SAB4503785), c-Abl (Sigma #cat A5844), anti-c-Abl (Santa Cruz Biotechnology, Dallas, TX, USA; #cat K-12), anti-c-Abl (Santa Cruz Biotechnology, #cat 24-11), anti-phospho-c-Abl (Tyr 412) (Sigma, #cat C5240), anti-PLC-γ (Cell Signaling, Danvers, MA, USA; #cat 2822), anti-phospho-PLC-γ (Tyr 783) (Cell Signaling, #cat 2821), anti-AKT (Cell Signaling, #cat 9272), anti-phospho-AKT (Ser 473) (Cell Signaling, #cat 9291), anti-ERK (Cell Signaling, #cat 9102), anti-phospho-MAPK (Erk1/2; p42/44; Thr202/Tyr204) (Cell Signaling, #cat 9101), anti-Flag (Sigma, #cat F3040), anti-GAPDH (Santa Cruz, #cat 6C5), β-III Tubulin (Sigma, #cat T8578), anti-MAP2 (Sigma, #cat M3696), anti-phospho-CrkII (Tyr221) (Cell Signaling, #cat 3491), anti-CrkII (Cell Signaling, #cat 3492), Secondary Antibody: Goat anti-Mouse IgG (H + L) and Goat anti-Rabbit IgG (H + L), HRP (Invitrogen, Carlsbad, CA, USA, #cat 31430 and #cat 31460) and secondary antibody Alexa-555, Alexa-488, Alexa-633 and Alexa 547 (1:1000) (Invitrogen, Carlsbad, CA, USA).

DC-1 cells were prepulsed with 5 μM MCC_88-103 overnight at 37 °C and washed before the assay. 2 × 10⁶ mPD-1-deleted 2D12 cells transduced with hPD-1 were stimulated with 2 × 10⁶ DC-1 cells not transduced or transduced with hPD-L1. The cells were lysed with the lysis buffer (50 mM Tris-HCl, 50 mM NaCl, and 5 mM EDTA) containing 1% NP-40. Whole cell lysates (WCLs) or those immunoprecipitated by anti-GFP (MBL International, D153-11, RRID:AB_2893312) were blotted with anti-GFP (1:5000, Miltenyi Biotec, 130-091-833, RRID:AB_247003), anti-mSHP1 (1:500, Santa Cruz Biotechnology, sc-287, RRID:AB_2173829), anti-mSHP2 (1:1000, Santa Cruz Biotechnology Inc., sc-7384, RRID:AB_628252), anti-PLCγ (1:1000, Cell Signaling Technology, 5690, RRID:AB_10691383), anti-pPLCγ (1:1000, Cell Signaling Technology, 8713, RRID:AB_10890863), anti-Akt (1:2000, Cell Signaling Technology, 4691, RRID:AB_915783), anti-pAkt (1:1000, Cell Signaling Technology, 4060, RRID:AB_2315049), anti-Erk (1:1000, Cell Signaling Technology, 4695, RRID:AB_390779), or anti-pErk (1:1000, Cell Signaling Technology, 4370, RRID:AB_2315112) as a first antibody and HRP-anti-rabbit IgG polyclonal Abs (1:10,000, Cell Signaling Technology, 7074, RRID:AB_2099233) as a second one. Each intensity of band was calculated by ImageJ (RRID:SCR_003070).

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).

Antibody detecting anti-Na,K-ATPase α1 subunit was purchased from Upstate (Billerica, MA). Anti-PLC-γ, -actin, -His, -cytochrome c, -caspase-9, -cleaved caspase-3, -cleaved caspase-7, -cleaved caspase-9, -cleaved PARP, -Flag, and -Apaf-1 antibodies were from Cell Signaling Technology (Boston, MA). Anti-EGFR, and -GFP antibodies were from Santa Cruz (Santa Cruz, CA). Anti-TCTP-specific antibody was from LabFrontier (Korea). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was from Molecular Probe (Carlsbad, MO). Etoposide, Taxol (paclitaxel), Ac-LEHD and Ac-DEVD were from Calbiochem (San Diego, CA). Bovine serum albumin (BSA), dATP, cytochrome c, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were from Sigma (St Louis, MO). Anti-OxPhos Complex IV (COX IV) antibody was from Invitrogen (Carlsbad, CA). Purified WD Repeat (WDR) protein was from Abnova Corporation (Taiwan).

Reagents: picrotoxin and strychnine were purchased from Sigma (St. Louis, MO, USA) and were dissolved in ethanol (96%, Sigma) and water, respectively. Tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel) was dissolved in water.
Primary Antibodies: anti-pTrkA^Y490 (Cell Signalling, Danvers, MA, USA, 9141), anti-Trk (Santa Cruz, Santa Cruz, CA, USA, sc7268), anti-pShc^Y317 (Cell Signalling, 2434), anti-ShcC (Cell Signalling, 2431), anti-pPLC-γ ^Y783 (Cell Signalling, 2821), anti PLC-γ (Cell Signalling, 14008), anti-pMAPK^Y202/Y204 (Cell Signalling, 9101), anti-MAPK (Cell Signalling, 9102), anti-pPI3K^Y458/Y199 (Cell Signalling, 4228), anti-pAKT^S473 (Cell Signalling, 4051), anti-AKT (11E7) (Cell Signalling, 4685), anti-pCREB^S133 (Millipore, Temecula, CA, USA, 06-519), anti-BDNF (Millipore, MABN110), anti-c-Fos (Cell Signalling, Rb Mab 2250S), anti-ChAT (Millipore, AB144P), and anti-NeuN (ABCAM, city, state abbrev if USA, country ab177487), anti-MAP2 (Cell Signalling, 4542; Millipore, MAB3418). Secondary antibodies: anti mouse-HRP and anti rabbit-HRP (PerkinElmer, Waltham, MA, USA); Donkey anti mouse-Alexa546, donkey anti rabbit-Alexa488 (Life Technologies, Carlsbad, CA, USA).