Anti phosphorylated c met

Cells growing in Petri dishes were collected and lysed in cell lysis buffer (Cell Signaling Technology, Boston MA USA) containing 20 mmol/L Tris-HCL (PH7.5), 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGDA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO4, 1 μg/ml leupeptin, and 1 mmol/l phenylmethane sulphonyl fluoride. Membranes were incubated overnight at 4°C with specific antibodies in TBS-T (TBS-0.1% Tween20) and subsequently for one hour with the appropriate horseradish-peroxidase-conjugated anti-rabbit/mouse secondary antibody. The following mouse monoclonal antibodies were used: Anti-CyclinB1 (Santa Cruz Biotechnology, Dallas Texas USA), anti-Caspase-8, anti-β-actin (both from Cell Signaling Technology, Boston MA USA). The following rabbit polyclonal antibodies were used: anti-Caspase-3, anti-PARP, anti-Bid, anti-Bad, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Mcl-1 (all from Cell Signaling Technology), anti-c-MET, anti-phosphorylated c-MET (Tyr1349 and Tyr1234/1235). For Caspase-3/7 activity assays the Apo-ONE® Reagent kit (Promega, Madison WI USA) was used according to the instructions of the manufacturer. Actin was used for control of appropriate protein load for each membrane. In figures, one actin loading control has been exemplarily shown.

Whole liver tissues were homogenized in lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, phosphatase (Pierce, Thermo Fisher Scientific, A32957), and protease inhibitors (MilliporeSigma, P8340). Protein concentration was measured with the BCA method (Thermo Fisher Scientific, 23225). Approximately 15–40 μg of protein was used for liver immunoblots. For plasma samples, 1 μL of plasma was mixed directly with 15 μL of Laemmli buffer with reducing reagent added (NuPAGE Sample Reducing Agent, NP0004). Lysates were then subjected to immunoblotting with the indicated antibodies: anti-HGFAC (R&D Systems, Bio-Techne, AF1715), anti–β-actin (Cell Signaling Technology, 4970S), anti–phosphorylated c-MET (Cell Signaling Technology 3077), anti–total c-MET (Cell Signaling Technology 3127), anti-PPARγ (Cell Signaling Technology 2435), anti-PDHA1 (phospho-S293) (Abcam, ab92696), anti-PDH (Cell Signaling Technology 3205), anti-p85 (Upstate, 06-496), and anti-PCNA (Cell Signaling Technology, 2586). Quantification of blots was performed with a ChemiDoc XP (Bio-Rad) and Image Lab software v6.0. For loading normalization, whole-lane protein was quantified using Bio-Rad Stain-Free technology.