Igf2bp2

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.

Total protein was obtained from cell samples with the RIPA buffer (Beyotime, China) supplemented with protease inhibitor cocktail tablet (Roche, Switzerland). Western blot was carried out with rabbit antibodies targeting IGF2BP2 (1:5000, Abcam, UK), cyclinD1 (1:1000, Abways, china), AKT (1:1000, Abways, china), p-AKT (1:1000, Abways, china), and GAPDH (1:5000, ZENbio, China) antibodies. HRP-conjugated IgG (1:5000, Beyotime, China) was utilized as secondary antibodies. The ECL-Plus reagent (Millipore, USA) was utilized for development. GAPDH was used for normalization.

Exosome, cell, and liver tissue protein concentrations were measured with the Bradford assay. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidine difluoride (PVDF) membranes. Membranes were incubated with primary antibodies at 4°C overnight in a buffer containing 5% skim milk, and then with a horseradish peroxidase (HRP)-conjugated secondary antibody at 37°C for 2 h. For exosome validation, primary antibodies against TSG101 (1:1000), CD9 (1:1000), MHC-1 (1:1000), and Cytochrome C (1:1000) were purchased from Abcam (Cambridge, UK). For cell migration and invasion assays, primary antibodies against CXCR4(1:500) and MMP-9 (1:500) were purchased from Boster (Wuhan, China). For liver pre-metastatic niche formation assays, primary antibodies against CD11b (1:1000), S100A8(1:1000), and S100A9 (1:1000) were purchased from Santa Cruz (CA, USA). For MS-identified candidates, primary antibodies against FRA2 (1:1000), IGF2BP2 (1:1000), and S100A11 (1:1000) were purchased from Abcam (Cambridge, UK). Selenium binding protein 1(SBP1, 1:500), CKAP4 (1:500), and aldolase C (ALDOC,1:500) were purchased from Bioss(Beijing, China). Protein-band densities were analyzed quantitatively using ImageJ software (NIH, USA).