Protease and phosphatase inhibitors tablets

293T cells were transfected with the construct encoding c-MET-C-GFPSpark. Cells were stimulated with either HGF WT or HGF 4Cys-4Ala as indicated following serum starvation. Cells were collected at indicated time points and washed with ice-cold PBS, lysed in ice-cold buffer containing 10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 20 mM NEM (N-ethylmaleimide) supplemented with protease and phosphatase inhibitors tablets (Thermo Fisher Scientific). The lysates were cleared by centrifugation at 10,000 × g at 4 °C for 20 min, followed by preclearance using protein G-beads (protein G from Thermo Fisher). 500 µg of each precleared lysate was incubated with anti-MET antibody and protein-G beads for overnight at 4 °C with rotation. The samples were then washed three times with the lysis buffer and eluted with SDS-gel loading buffer (with reducing agent added). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

HEK 293 T cells were obtained from ATCC and maintained in a humidified atmosphere at 5% CO₂ in Dulbecco’s Modified Eagle’s (DMEM) complete medium (Corning) supplemented with 10% fetal bovine serum (FBS; Seradigm) in 37 °C. Plasmid transfections were done with TransIT-LT1 (Mirus Bio) per the manufacturer’s instructions. Briefly, cell extracts were generated on ice in EBC buffer, 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP40, 1 mM DTT, and protease and phosphatase inhibitors tablets (Thermo Fisher Scientific). Extracted proteins were quantified using the PierceTM BCA Protein assay kit (Thermo Fisher). Proteins were separated by SDS acrylamide gel electrophoresis and transferred to IMMOBILON-FL 26 PVDF membrane (Millipore) probed with the indicated antibodies and visualized either by chemiluminescence (according to the manufacturer’s instructions) or using a LiCor Odyssey infrared imaging system. Western blot was conducted as described previously^{10 (link)}. Primary antibodies used for western blot are HA (Cat# 902302; 1:1000 dilution) from Biolegend and M2 FLAG (Cat# F1804; 1:1000 dilution) antibody from Sigma. Secondary antibodies used are IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LiCor) and IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LiCor). The study used a primary antibody against β-actin (Cat# A1978 from Sigma) for internal protein control.

NuPAGE® MOPS SDS Running Buffer (20X), (#NP0001–02), NuPAGE® MES SDS Running Buffer (20X), (#NP0002–02), NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.0 mmX15well, (#NP0323BOX), NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.0 mmX26well, (#WG1403BOX) and PierceTM BCA Protein Assay Kit (#23225) and ProLong Diamond antifade mountant with or without DAPI (#P36962 or #P36961) were from Thermo Scientific. Western Lightning Plus ECL (#NEL105001EA) was from PerkinElmer. Immobilon®-FL Transfer membrane 0.45 um, Polyvinylidene Difluoride (PVDF) membrane (#ISEQ00010) was from Merck Millipore. HyBlot CL films (#E3012) were from Denville Scientific, Inc. Non-fat dry milk (#M0841) was from Lab Scientific. Protease and Phosphatase inhibitors tablets (#88669) were from Thermo Scientific. Dynabeads Protein G was from Novex (Life Technologies, #10004D). Ponceau S Solution (#P7170) was from Sigma-Aldrich. OCT compound (#23–730-571) and microscope slides (# 12–550-15) were from Fisher Scientific. Liquid blocker pap pen (#71310) was from Electron Microscopy Sciences (EMS).