Ac002

The cells were lysed in RIPA buffer (Sparkjade, Jinan, China) supplemented with protease inhibitor cocktail (Servicebio, Wuhan, China) and phosphatase inhibitors A and B on ice for 30 min. The lysates were centrifuged for 15 min at 12,000 rpm, and their protein content was measured. Equal amounts of protein per sample were resolved on 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% BSA in TBST for 2 h at room temperature, the membranes were incubated overnight with antibodies targeting GAPDH (Abclonal, Wuhan, China, AC002), RRS1 (Abcam, Cambridge, UK, AB188161), AEG-1 (Abcam, Cambridge, UK, ab227981), ABCG2 (ZenBio, Research Triangle Park, NC, USA, R26465), MDR1 (Proteintech, Wuhan, China, 22336-1-AP), ERK (ZenBio, NC, USA, 340373), p-ERK (ZenBio, NC, USA, 340767), BAX (CST, Danvers, MA, USA, #89477), Bcl-2 (CST, Danvers, MA, USA, #3498), BAD (Abcam, Cambridge, UK, ab32445), p-BAD (Abcam, Cambridge, UK, ab129192) and ubiquitin (Proteintech, Wuhan, China, 10201-2-AP) at 4 °C. The membranes were washed thrice with TBST and then incubated with HRP-conjugated secondary antibody (1:1000; Bioss, Beijing, China, bs-0295G-HRP), followed by three more washes with TBST. The positive bands were visualized via ECL (MDBio, Taiwan, China).

Total proteins were extracted from CRC cell lines by using RIPA lysis buffer (Beyotime, China) supplemented with PMSF (Beyotime, China) and phosphatase inhibitor (Roche, Switzerland) and then quantified using BCA Protein Assay Kit (Beyotime, China). Nuclear and cytoplasmic protein were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Absin, China) according to the protocol provided by the manufacturer. Protein was electrophoresed through 10% SDS polyacrylamide gels and then transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk for 2 h and incubated with primary antibodies at 4 °C overnight. Membranes were then incubated with second antibodies labeled with HRP at room temperature for 2 h on the following day and the signal was detected using an ECL kit (Beyotime, China). The following primary antibodies were used: JNK (Abcam # ab179461), P-JNK (Abcam # ab124956), c-Jun (CST# 9165), P-c-Jun (Abcam#ab32385), and MMP7 (Abcam#ab205525). β-actin (Abclonal#AC026), GAPDH (Abclonal#AC002) or PCNA (Abcam#ab29) was used as reference gene.