Irdye 680 conjugated goat polyclonal anti rabbit igg

Cells were lysed in 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, and 150 mM NaCl, containing an EDTA-free protease inhibitor tablet (Roche, Mississauga, ON, Canada), 1 mM EGTA, 1 mM Na₃VO₄, 1 mM sodium pyrophosphate, 1 μg/mL aprotinin, and 1 μg/mL pepstatin. The protein concentrations of lysates were determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and 25 µg total protein was loaded per lane. Western blotting was performed using affinity-purified rabbit polyclonal NGF antibody (MC-51), anti-TrkA rabbit polyclonal antibody (Chemicon, Mississauga, ON, Canada), anti-sortilin rabbit monoclonal antibody (Alomone Labs Ltd., Jerusalem, Israel), anti-p75NTR rabbit polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY, USA), anti-phosphorylated anti-extracellular signal-regulated kinase (ERK) 1/2, Thr202/Tyr204), and total ERK (Cell Signaling Technology, Inc., Mississauga, ON, Canada), anti-phosphorylated Akt (Ser473) and total Akt, or monoclonal mouse anti-β-actin antibody (clone AC-51, Sigma, Oakville, ON, Canada). The secondary antibodies used were IRDye^® 680 conjugated goat polyclonal anti-rabbit IgG or IRDye^® 800 conjugated polyclonal anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA). The membranes were scanned at 680 nm and/or 800 nm using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA). PKC inhibitor Gö6983, PI3K inhibitor LY294202, and MEK1/2 inhibitor U0126 were purchased from Calbiochem (San Diego, CA, USA). AT1 receptor blocker candesartan was from AstraZeneca. Neutralizing anti-IGFR1 antibody, MAB 391, was bought from R & D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium/F12 (DMEM/F12) was from Life Technologies (Carlsbad, CA, USA). Nu serumTM and ITS+premix were obtained from BD Biosciences (Bedford, MA, USA). Anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-AKT (Ser473), anti-ERK1/2, and anti-AKT antibodies were from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies [IRDye 800CW Conjugated Goat (polyclonal) anti-mouse IgG and IRDye 680 Conjugated Goat (polyclonal) anti-rabbit IgG] were products of LI-COR Biosciences (Lincoln, NE, USA). High-capacity cDNA reverse transcription kit was from Applied Biosystems. LightCycler® 480 SYBR Green I Master was purchased from Roche Applied Science. Trizol reagent and all other reagents came from Sigma-Aldrich (St. Louis, MO, USA).