Pierce immunoprecipitation lysis buffer

For immunoprecipitation, at 48 h after plasmid transfection, HEK293T cells were washed twice with ice-cold phosphate-buffered saline and then lysed in Pierce immunoprecipitation lysis buffer (Thermo Fisher Scientific; #87787) with protease inhibitor for 20 min at 4 °C. Crude lysates were cleared by centrifugation at 20,000×g at 4 °C for 10 min, and the supernatant was incubated with GFP-Trap (ChromoTek, Hauppauge, NY, USA) for 2 h at 4 °C. The immunoprecipitates were extensively washed three times with lysis buffer and eluted with 2× sodium dodecyl sulfate–polyacrylamide gel electrophoresis loading buffer by boiling for 10 min. For immunoblotting, whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (#R0278, Sigma-Aldrich, St. Louis, MO, USA) containing protease and phosphatase inhibitors (Thermo Fisher Scientific; 78,440). To induce protein phosphorylation, cells were treated with 20 ng/ml recombinant human NRG-1β (R&D Systems, Minneapolis, MN, USA. #396HB) for 30 min before they were harvested. Samples were subjected to SDS–PAGE and immunoblotting using standard procedures.

Cells were washed on the plate with PBS and lysed in Pierce immunoprecipitation lysis buffer (Thermo Fisher Scientific, PI87787) supplemented with Pierce Halt protease and phosphatase inhibitor cocktail (no. 78443). Immunoprecipitation was performed by incubating protein lysates with antibody-coupled magnetic beads (anti-HA magnetic beads [Cell Signaling Technology, 11846S] and anti-myc magnetic beads [Thermo Fisher Scientific, PI88842]) overnight at 4°C with rotation followed by three washes with lysis buffer. Samples were either brought for mass spectrometry analysis or eluted with SDS-PAGE gel loading buffer (Thermo Fisher Scientific, 50194470) for Western blot analysis; otherwise, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail for direct Western blot analysis.

Fibroblasts were lysed in ice-cold Pierce immunoprecipitation lysis buffer (ThermoFisher Scientific) supplemented with EDTA-free protease and phosphatase inhibitors (ThermoFisher Scientific). Association of proteins with the 5′ mRNA cap was determined by first incubating lysates with m⁷GTP-bound sepharose beads (Jena Bioscience), followed by Western blot to assess the association of relevant proteins.