Immobilob western chemiluminescent hrp substrate

Cell lysates were prepared with RIPA (Beyotime, Shanghai, China) buffer to isolate total cellular protein. The proteins were then resolved by SDS-PAGE using a 10% gel, and transferred wet to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% BSA and the following primary antibodies applied overnight at 4 °C: ALK (#3633, D5F3R, 1:2000), p-ALK (#3341, 1:1000), PI3K (#4257, 1:1000), p-PI3K (#4428, 1:1000), AKT (#4691, 1:1000), p-AKT (#5012, 1:2000), mTOR (#2983, 1:1000), p-MTOR (#5536, 1:1000), JAK3 (#8827, 1:1000), p-JAK3 (#5031, 1:1000), STAT3 (#12640, 1:1000), p-STAT3 (#8204,1:2000), MEK1/2 (#8727, 1:1000), p-MEK1/2 (#9154, 1:1000), ERK1/2 (#4695, 1:1000), p-ERK1/2 (#8201, 1:1000), XIAP (#2045, 1:1000), Bac-xl (#2764, 1:1000), Bid (#2002, 1:1000)), Bax (#5023, 1:1000), Cleaved Caspase-3 (#9664, 1:1000), and GAPDH (#5174, 1:1000) (all from Cell Signaling Technology, Beverly, MA, USA). On the next day, blots were incubated with secondary anti-rabbit (1:3000, Cell Signaling Technology, Beverly, MA, USA) at room temperature for 1 h. Finally, protein bands were detected with Immobilob™ Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA), using a fully automated chemiluminescence imaging analysis system (Tanon 5200, Shanghai, China).

Total proteins from human breast cancer cells were extracted using RIPA buffer (P0013C, Beyotime, China) containing 1% phosphatase inhibitor, 1% PMSF and 0.1% protease inhibitor. Total proteins were separated using 10% sodium dodecyl sulfate‐polyacrylamidegel electrophoresis (SDS‐PAGE) and transferred to PVDF (polyvinylidene fluoride) membranes (Millipore, USA). The membranes were placed in Tris‐buffered saline containing 0.1% Tween 20 (TBST) and 5% bovine serum albumin (BSA) for 2 h before the primary antibody was placed and left overnight at 4 °C. The primary antibody stock solution used was shown below. IGF2BP2 (1:1000, Abcam, UK, ab128175), GAPDH (1:1000, Beyotime, China, AF1186), CDK6 (1:1000, Protein‐tech, China, 14052‐1‐AP), EIF4A1 (1:500, Abcam, UK, ab31217), TET1 (1:1000, Affinity, USA, DF6428), TET2 (1:1000, Affinity, USA, DF12089), TET3 (1:1000, Affinity, USA, DF13335), CDK4 (1:1000, Protein‐tech, China, 11026‐1‐AP), CCND1 (1:1000, Protein‐tech, China, 26939‐1‐AP), pRb (1:500, Affinity, USA, AF0030). PVDF membranes were washed three times with TBST and incubated with secondary antibody (1:5000, Cell Signaling Technology, USA, 7074P2) for 2 h at room temperature. Immobilob Western Chemiluminescent HRP Substrate (Millipore, USA) was used to detect the expression level of the target protein.

The total protein of breast cancer cells was exacted with RIPA buffer and separated by 10% SDS-PAGE, then electransferred onto a PVDF membrane (Bio-Rad, USA). The membranes were blocked with 5% skimmed milk powder and incubated with primary antibodies against METTL3 (1:1000, Abcam, USA, ab195352), CDK1 (1:1000, Proteintech, USA, 19532-1-AP), β-actin (Proteintech, China, 20536-1-AP) at 4 °C overnight and then incubated with secondary antibodies (1:5000) (Cell Signaling Technology, USA, 7074P2) at room temperature for 2h. Finally, the bands were examined by Immobilob™ Western Chemiluminescent HRP Substrate (Millipore, USA).