Wbkis0100

Hundred micrograms of protein per lane was subjected to 10% SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk (D8340, Solarbio, Beijing, China) and incubated with primary antibodies against TOM70 (1:1000 dilution, ab89624, Abcam, Shanghai, China; 1:1000 dilution, 14528-1-AP, Proteintech, Wuhan, Hubei, China), TOM40 (1:2000 dilution,18409-1-AP, Proteintech, Wuhan, Hubei, China), TOM22 (1:500 dilution, 11278-1-AP, Proteintech, Wuhan, Hubei, China), TOM20 (1:2000 dilution, 11802-1-AP, Proteintech, Wuhan, Hubei, China), β-actin (1:2000 dilution, 20536-1-AP, Proteintech, Wuhan, Hubei, China), or GAPDH (1:50000 dilution, 60004-1-AP, Proteintech Wuhan, Hubei, China) overnight at 4°C, followed by incubation with secondary species-specific HRP-coupled antibodies (1:5000 dilution, E-AB-1034, Elabscience Wuhan, Hubei, China) for 1 h at room temperature. Proteins were visualized using an ECL chemiluminescence kit (WBKIS0100, Millipore, Darmstadt, Germany) and a chemiluminescence imaging system (ProteinSimple, ChenQ, CA, USA). Bands were quantified using ImageJ software.

Western blotting was performed as described in our previous study 40 (link). Briefly, total protein lysates were prepared from cells with SDS buffer with proteinase. A BCA assay (P1002, Beyotime, Shanghai, China) was performed to quantify the protein concentrations. The obtained protein samples were resolved using 10% SDS-PAGE and transferred onto a PVDF membrane (#ISEQ00010, Millipore, Temecula, CA, USA). After being blocked with 5% fat-free milk, the membranes were cut into strips with molecular weight cut in half in-between the strips. Then, the strips were incubated overnight at 4°C with primary antibodies (the antibodies information was listed in Table S7). After incubation with secondary antibodies, the immunoreactive bands were visualized using an enhanced chemiluminescence reagent (WBKIS0100, Millipore, Temecula, CA, USA). The densities of the immunoreactive bands were determined by Quantity One 1-D Analysis Software, (RRID:SCR_014280).