Mini gel tank chamber system

Cells were lysed in RIPA buffer (Cat# 89900, Thermo Fisher Scientific, Waltham, MA, USA) containing 1% protease and phosphatase inhibitors (Cat# 78442, Thermo Fisher Scientific). Protein concentrations were quantified using a BCA Protein Assay Kit (Cat# 23250, Thermo Fisher Scientific). A quantity of 20 µg of protein samples was loaded onto 4–12% NUPAGE Bis-Tris gels (Cat# NP0322BOX; Thermo Fisher Scientific) using a Mini Gel Tank chamber system (Invitrogen), and the proteins were transferred to Amersham™ Protran™ Premium 0.45 µm NC membranes (Cat# GE10600003, Cytiva, Freiburg, Germany). After blocking with 5% milk, primary antibodies (listed in Table S5) were used according to the manufacturer’s requirements and incubated overnight at 4 °C. HRP-conjugated anti-rabbit or anti-mouse IgG was used as a secondary antibody. Signal quantification was performed with an Amersham Imager 600 (Pittsburgh, PA, USA) and SignalFire™ ECL reagent (Cat# 6883S, Cell Signaling Technology Europe, Leiden, The Netherlands). We individually performed all Western blot assays 3 times.

The total protein was lysed using the Pierce^TM RIPA Buffer (Thermo Scientific, 89900, Rockford, IL, USA) mixed with Halt^TM Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78442, Rockford, IL, USA). The quantitation of proteins was carried out using a Pierce^TM BCA Protein Assay Kit (Thermo scientific, 23227, Rockford, IL, USA). Protein samples were prepared by mixing an equal amount of protein with LDS Sample Buffer (Novex^TM by life technologies, B0008, Carlsbad, CA, USA) and Sample Reducing Agent (Novex^TM by life technologies, B0009) before denaturation. Gel electrophoresis was performed using a Mini Gel Tank chamber system (Invitrogen) with Bolt^TM 4–12% Bis-Tris gel (Invitrogen, NW04122BOX, Carlsbad, CA, USA), followed by transferring the protein to the Nitrocellulose Blotting membrane (Amersham^TM, 10600003, Germany). 5% BSA or nonfat milk was used to block nonspecific antigens, and primary antibodies were incubated overnight at 4 °C, including phospho-p44/42 ERK1/2 (CST, 4370, RRID: AB_2315112), p44/42 ERK (CST, 4695, RRID: AB_390779) and GAPDH (CST, 5174, RRID: AB_10622025). HRP-linked Anti Rabbit IgG (CST, 7074, RRID: AB_2099233) was used as secondary antibody. Examination of the signal was performed using Amersham Imager 600 (Pittsburgh, PA, USA) with SignalFire™ ECL Reagent (CST, 6883S, Frankfurt, Germany).

Western blot analysis was performed as described [37 (link)]. In brief, cells were lysed in RIPA buffer (Cat. #89,900, Thermo Fisher Scientific) containing protease and phosphatase inhibitor (Cat. #78,442, Thermo Fisher Scientific), and the protein concentration was determined photometrically using a BCA Protein Assay Kit (Cat. #23,250, Thermo Fisher Scientific). Equal amounts of total protein were separated on 4–12% NUPAGE Bis–Tris gels (Cat. #NP0322BOX; Thermo Fisher Scientific) using the Mini Gel Tank chamber system (Cat. #A25977, Invitrogen), and proteins were transferred to a nitrocellulose membrane (Cat. #GE10600003, Sigma Aldrich). Membranes were then blocked in blocking buffer (Cat. # A0830.1000, AppliChem GmbH) for 1 h at room temperature and incubated with appropriate primary antibodies (Supplementary Table S3) overnight at 4 °C. HRP-linked anti-rabbit IgG or anti-mouse IgG were used as the secondary antibodies. Signal detection was performed using an Amersham Imager 600 (Pittsburgh, PA, USA) with SignalFire™ ECL Reagent (Cat. #6883S, Cell Signaling Technology).