Electrochemiluminescence reagents

For immunoblotting, RIPA buffer supplemented with phosphatase and protease inhibitors (#B15003 and #B14002, Bimake, Texas, America) was used to lyse cells and tissues. After quantification using a bicinchoninic acid kit (#20201ES90, Yeasen, Shanghai, China), equal quantities of proteins were separated and then transferred to PVDF membranes (#IPVH00010, Millipore, Bedford, MA, USA). After incubation with antibodies, the protein signals were detected using electrochemiluminescence reagents (#36208ES76, Yeasen, Shanghai, China). For IP assays, cellular extracts were incubated with anti-HA (#B26202, Bimake, Texas, America) or anti-Flag beads (#B26102, Biotool, Beijing, China) at 4 °C, followed by immunoblotting. The information on antibodies is listed in Supplementary Table S4.

Tissues and cells were lysed with RIPA buffer containing protease inhibitor (Bimake, #B14002) and phosphatase inhibitor (Bimake, #B15003). Bicinchoninic acid (BCA) protein assay kit (Yeasen, #20201ES90) was used to detect protein concentration. Equal quantity of proteins was separated by SDS‐PAGE and transferred to PVDF membrane. The membrane was blocked with 5% bovine serum albumin (Yeasen, #36101ES80) for 1–3 h at room temperature, and subsequently incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1–3 h at room temperature. Next, the electrochemiluminescence reagents (Yeasen, #36208ES76) were used to detect the antibody signals, and quantitative analysis of the developed results was performed by ImageJ software using vinculin as an internal reference to calculate the relative expression levels of target protein. Primary antibodies of IMPA1(Abcam, #ab184165), Vinculin (Sigma, #V9131), and HA (CST, #3724S) were used in this study. Other antibody information is shown in Table S5.