Immobilon fl 26 pvdf membrane

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.

For in vivo stimulation experiments, cells were grown for 36 h and then stimulated with HGF WT and HGF 4Cys-4Ala where indicated (100 ng mL⁻¹), washed with PBS, and lysed. 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.
For immunoprecipitation, endogenous c-MET was immunoprecipitated on c-MET antibody-bound beads (Dynabeads Protein G from Thermo Fisher) and Flag-tagged HGF WT and HGF 4Cys-4Ala were in-vitro translated (T_NT quick coupled Transcription/Translation system, Promega) and were incubated with the bead bound c-MET for 4 h at 4 °C. Beads were then washed and proteins resolved by SDS-PAGE and analyzed by western blotting as above.