Immune blot pvdf membranes

Total proteins were extracted using GTEN buffer as described by Du et al. (2021). For Co‐IP assays, for each 1.6 ml of total protein extraction a 12 µl of GFP‐trap_A beads (Chromotek) were added, and the mixtures were incubated for 2 hr with gentle shaking at 4 °C before proteins were spun down. The beads were washed with protein extraction buffer five times before being boiled in protein loading buffer. For western blot assays, proteins were separated on 10% sodium dodecyl sulphate (SDS)‐polyacrylamide gels and subsequently transferred to Immune‐Blot PVDF membranes (Roche). The membranes were blocked in 10 ml of blocking buffer (TBST; Tris‐buffered saline pH 7.2, 0.05% Tween 20 [Sigma]) containing 5% of nonfat milk, with gentle shaking for 2 hr at room temperature. Subsequently, membranes were incubated with anti‐GFP (#AE012, ABclonal), anti‐myc (#AE010, ABclonal), or anti‐pERK antibody at the appropriate dilution for 1.5–2 hr with gentle shaking at room temperature. Then the membranes were washed five times and incubated with the secondary antibodies horseradish peroxidase‐conjugated goat anti‐rabbit IgG (H + L) antibody (#AS014, ABclonal) for another 1.5–2 hr. After this incubation, membranes were washed with TBST five times before proteins were detected (eECL western blot kit; CWBio).

Proteins were separated on SDS‐PAGE gels (10%), before being transferred to Immune‐Blot PVDF membranes (Roche). Subsequently, the membranes were incubated in blocking buffer for 2 h at 4°C. After blocking, the membranes were incubated with anti‐His (#AE003, ABclonal), anti‐GFP (#AE011, ABclonal), anti‐GST (#AE006, ABclonal), anti‐MBP (#AE016, ABclonal), anti‐myc (#AE009, ABclonal), anti‐actin (#AC009, ABclonal) antibodies for 2 h at 4°C. The membranes were subsequently washed with blocking buffer for three times, before being incubated with the secondary antibody HRP goat anti‐rabbit IgG (H + L) antibody (#AS014, ABclonal), or HRP goat anti‐mouse IgG (H + L) antibody (#AS003, ABclonal). Finally, the membranes were washed with TBST and they were developed using the eECL western blot kit (CWBIO, Beijing, China).