Whole cell lysis assay kit

Proteins were collected from cells using the Whole Cell Lysis Assay Kit (KGP250, KeyGen, Nanjing, China). The protein concentration was determined using the bicinchoninic acid (BCA) method using the BCA Protein Quantitation Assay Kit (KeyGen, Nanjing, China). Protein was electrophoretically separated by 10% or 15% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were blocked for 1 hour with 5% bovine serum albumin (BSA) in TBS-T and incubated with specific primary antibodies overnight at 4° C followed by incubation with rabbit or mouse radish peroxidase-coupled secondary antibodies for 1 hour. Antibody binding was detected using the enhanced chemiluminescence reagent (Millipore, Billerica, MA). The antibodies used in this study were as follows: ALKBH5 (ab195377, Abcam), H3K27ac (#8173, Cell Signaling Technology [CST]), cyclin D1 (#2978, CST), cyclin E1 (ab33911, Abcam), c-Myc (#5605, CST), caspase-3 (19677-1-AP, Proteintech), BCL-2 (12789-1-AP, Proteintech), MMP2 (#87809, CST), MMP7 (#71031, CST), MMP9 (#13667, CST), E-cadherin (#3195, CST), N-cadherin (#13116, CST), vimentin (#5741, CST), β-catenin (#8480, CST), Snail (ab53519, Abcam), Slug (ab27568, Abcam), β-actin (ab8227, Abcam), and GAPDH (#8884, CST).

Whole Cell Lysis Assay kit (Keygen, KGP2100) was used to extract proteins from cells and small extracellular vesicles. Total protein concentration was analyzed using the BCA Protein Quantitation Assay kit (Keygen, KGPBCA). Western blotting was performed as previously described [19 (link)]. Antibodies used were as follows: TSG101 (1 : 1000, AF8259, Beyotime), CD63 (1 : 1000, AF1471, Beyotime), CD81 (1 : 1000, AF2428, Beyotime), P21 (1 : 1000, 10355-1-AP, Proteintech), BAX (1 : 1000, 50599-2-Ig, Proteintech), Bcl-2 (1 : 1000, 26593-1-AP, Proteintech), phosphorylated-AKT (p-AKT) (1 : 1000, 28731-1-AP, Proteintech), AKT (1 : 1000, AF1789, Beyotime), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 1000, 10494-1-AP, Proteintech), and beta-actin (β-actin, 1 : 1000, 20536-1-AP, Proteintech). The bands were visualized by using enhanced chemiluminescence (Vazyme, E412-01) and analyzed with a gel documentation system. ImageJ was used for gray analysis of strips.

Total protein was extracted from the cells using whole cell lysis assay kit (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Equivalent amounts of proteins from each sample were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore), blocked in 5% fat-free milk for 1 h at room temperature, incubated with specific primary antibodies overnight at 4 °C with gentle shaking, and followed by detection with enhanced chemiluminescence system (SuperSignal West Femto trial kit, Pierce). Primary antibodies were as follows: E-cadherin (1:300, 3195, CST), Vimentin (1:1000, 5741, CST), NLRP3 (1:1000, ab16097, Abcam), and ACTB (1:5000, AT0001, CMCTAG).

A Whole Cell Lysis Assay Kit (KeyGen, China) was used to extract the total proteins, and a BCA protein assay kit (Beyotime, China) was then used to measure the protein concentration. Equal protein samples from each group were separated by 10% SDS–PAGE gels and then transferred to PVDF membranes (Millipore, USA). Subsequently, the membranes were blocked in 5% nonfat milk 2 h on a shaker at room temperature and incubated with primary antibodies overnight at 4°C. The membranes were then washed with TBST three times and incubated with secondary antibodies for 2 h at room temperature. Afterward, protein signals were visualized using an enhanced chemiluminescence system and analyzed using the ImageJ software. The loading control was β-actin.