Enhanced chemiluminescent kit

Proteins were extracted using radioimmunoprecipitation assay lysis buffer and protein concentration was measured using a Bicinchoninic Acid Protein Assay kit (both Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, 40 µg protein in each group were separated by 8, 10 or 12% SDS-PAGE and the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% skim milk or 1% bovine serum albumin (Biosharp, Hefei, China) at room temperature for 1 h, and then incubated with FOXC1 (1:1,000), N-cadherin (1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), Snail (1:1,000), Twist (1:1,000), β-catenin (1:1,000), p-β-catenin (1:1,000), c-myc (1:1,000) and GAPDH antibodies (1:1,000) overnight at 4°C. The PVDF membranes were rinsed and then incubated with corresponding horseradish peroxidase-labeled secondary antibodies (cat. no. A0208; 1:5,000; Beyotime Institute of Biotechnology) for 45 min at 37°C. Subsequently, the PVDF membranes were visualized using an enhanced chemiluminescent kit (Beyotime Institute of Biotechnology). The target bands were scanned and analyzed by Gel-Pro-Analyzer software version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Total protein of CRC cell lines was extracted by RIPA lysis buffer (Solarbio, Beijing, China) and the concentration was measured using BCA Protein assay kit (Beyotime, China). Equal amounts of total protein (30 μg) were separated by 12% SDS–PAGE and transferred onto PVDF membranes. After blocking with 5% BSA in TBST buffer for 2 h, the membranes were incubated with IGF1 antibody (1:1000 dilution; 133542; abcam, Cambridge, UK), or β-actin (1:5000 dilution; 66009-1-lg; Proteintech, Chicago, IL, USA) overnight at 4°C. Afterwards, the membranes were incubated with a corresponding HRP-labelled secondary antibody (1:5000, Lablead, Beijing, China) for 1 h at room temperature. Finally, the blots were visualized using an enhanced chemiluminescent kit (Beyotime, China). Quantification of the individual protein bands was performed by densitometry using ImageJ software.

The collected cells were lysed with radio immunoprecipitation assay buffer (Beyotime Institute of Biotechnology), and total protein concentration was determined using the bicinchoninic acid assay method. Protein samples (30 µg) were separated on a 10% SDS-PAGE gel and the proteins were then transferred to polyvinylidene fluoride membranes. The membranes were then blocked using 5% non-fat milk at 25°C for 1 h. Following this, membranes were incubated with primary antibodies against TRPV1 (1:2,000) and β-actin (1:1,000) at 4°C overnight. Membranes were then incubated with horseradish peroxidase (HRP)-anti-mouse (1:5,000) and HRP-anti-rabbit (1:5,000) antibodies at room temperature for 1 h. Proteins were then visualized using an enhanced chemiluminescent kit (Beyotime Institute of Biotechnology), and band intensities were densitometrically analyzed using a ChemiDoc XRS Protein Gel Imaging System (version 3.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Results are presented as the expression ratio of TRPV1 to β-actin.

Protein samples were extracted using RIPA lysis buffer (Servicebio, Wuhan, China), separated by SDS‐PAGE, and then transferred onto PVDF membrane (Bio‐Rad). The membranes were blocked by 5% skim milk and probed with the primary antibodies against multidrug resistance protein 1 (MRP1; ab170904; Abcam, Cambridge, MA, USA), MDR‐associated protein 1 (MRP1; ab263865; Abcam), E‐cadherin (ab40772; Abcam), N‐cadherin (ab18203; Abcam), PD‐L1 (ab243877; Abcam), β‐actin (ab8227; Abcam), TSG101 (ab125011; Abcam), CD63 (ab134045; Abcam), CD81 (ab109201; Abcam). The next day, the protein was incubated with the secondary antibody (ab205718; Abcam) for 2 h. The protein bands were visualized using an enhanced chemiluminescent kit (Beyotime, Shanghai, China).