Anti gapdh

Total proteins were isolated from cells or tissues using the RIPA lysis buffer (Beyotime, China) and subjected to 10% polyacrylamide gel electrophoresis. After transferring to PVDF membrane, blocking with 5% skim milk, incubation with primary antibodies and secondary antibodies, the proteins on the membrane were finally detected by ECL kit (EpiZyme, China) and imaging analysis system (BioRad, USA). The primary antibodies used in this study were as follows: anti-GAPDH (Bioworld, 1:10000), anti-CDK2 (Bioworld, 1:1000), anti-Cyclin E1 (Bioworld, 1:1000), anti-CDK4 (Bioworld, 1:1000), anti-Cyclin D1 (Bioworld, 1:1000), anti-p53 (Bioss, 1:500), anti-p21 (Bioworld, 1:500), anti-p16 (Bioss, 1:1000), anti-ubiquitin (Santa, 1:500), anti-FTO (Bioss, 1:1000), anti-YTHDF2 (Abcam, 1:1000).

A Western blot was used to detect protein levels of corresponding genes in CRC cells or nude mouse tumor tissues. In brief, total protein was extracted using a lysate mixture containing RIPA and protease inhibitors. After concentration determination, an equal amount of total proteins from each sample was used for polyacrylamide gel electrophoresis. After membrane transfer, blocking, antibody incubation, and coloration, protein bands on PVDF membranes were used for detection and analysis. Primary antibodies used in this study include anti-GAPDH (Bioworld, China), anti-SH3TC2 (Bioss, China), anti-CDK4 (Bioworld, China), anti-Cyclin D1 (Bioworld, China), and anti-YTHDF1 (Abcam, USA).

Proteins were isolated from atrial myocardium samples of patients and mice by M‐PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc) that contained protease inhibitors and phosphatase inhibitors. Primary anti‐GRP78 (sc‐376768, Santa Cruz), anti‐CHOP (#2895, Cell Signaling Technology), anti‐GAPDH (Bioworld, AP0063), anti‐phospho‐PERK (sc‐32577, Santa Cruz), anti‐PERK (sc‐13073, Santa Cruz), anti‐TGF‐β(Abcam, ab92486), anti‐α‐SMA(Abcam, ab32575), anti‐Collagen I (Abcam, ab34710) and anti‐Collagen III(Abcam, ab7778) were used for Western blotting experiments. Gel Imaging System (Tanon) and Image J software were used to image and analyse Western blotting bands.

Sample were added 100 μl of lysates on ice for 30 min. The cell and exosomes lysates were clarified by centrifugation at 12,000 rpm for 15 min, and the supernatants were collected. The protein concentration was measured with the QuantiPro BCA Assay Kit (KeyGen Biotech Co., Ltd., Shanghai, China). The protein concentration of each sample was measured with the QuantiPro BCA Assay Kit (KeyGen Biotech, Shanghai, China). 20 μg protein was applied to Western Blotting. The membranes were incubated overnight at 4°C with specific anti-CD63 (diluted 1: 200; Abcam, United States), anti-CD81 (diluted 1: 500; Abcam, United States), anti-CD40 (diluted 1: 1000; Bioss, China), anti-ALIX (diluted 1: 1000; Abbexa, United Kingdom), anti-RUNX2 (diluted 1: 500, SAB, United States), anti-BMP-2 (diluted 1: 500, Bioworld, United States), anti-OPN (diluted 1: 1000, Proteintech, United States), and anti-GAPDH (diluted 1: 5000, Bioworld, United States). Incubation with the secondary antibody (diluted 1: 500, ABclonal, China) lasted 1 h. The ECL luminescent solution was configured to collect the blotting results with a BIO-RAD gel imaging system, and the results were analyzed with Image Lab software.

The protein concentration of each sample was measured using a QuantiPro BCA Assay Kit (KeyGen Biotech, Shanghai, China). The membranes were then incubated overnight at 4 ºC with specific anti-CD63 (diluted 1: 200; Abcam, Cambridge, MA, USA), anti-CD81 (diluted 1: 500; Abcam), anti-CD40 (diluted 1: 1000; Bioss Antibodies, Woburn, MA, USA), anti-ALIX (diluted 1: 1000; Abbexa Ltd., Cambridge, UK), anti-RUNX2 (diluted 1: 500, SAB, USA), anti-BMP-2 (diluted 1: 500, Bioworld Technology, St Louis Park, MN, USA USA), anti-ALP (diluted 1: 1000, Abcam), anti-GJA1 (diluted 1: 1000, Bioworld), anti-AP3B1 (diluted 1: 300, Proteintech, Rosemont, IL, USA) and anti-GAPDH (diluted 1: 5000, Bioworld). Incubation with the secondary antibody (diluted 1:500, ABclonal, Woburn, MA, USA) lasted 1 h. The ECL luminescent solution was configured to collect the blotting results with a Bio-Rad gel imaging system (Bio-Rad, Hercules, CA, USA), and the results were analyzed using Image Lab software.

Cells were harvested and lysed respectively in IP Lysis Buffer (Thermo, USA) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). The protein samples were incubated with indicated antibody in 1 mL IP Lysis Buffer overnight at 4 °C, and then were precipitated with 20 μL Protein A/G Plus-agarose (Roche, Switzerland). After a brief centrifugation, the pellet was washed 3 times with IP Lysis Buffer. The lysates and immunoprecipitates were analyzed by immunoblotting.
Immunoblotting was performed using indicated primary antibodies: anti-MAGE-G1 (B-Bridge, USA), anti-GFP (Proteintech, USA), anti-GAP43 (Sigma-Aldrich, USA), anti-Neuron-specific III β-tubulin (Bioworld, China), anti-GAPDH (Bioworld, China), anti-active Caspase3 (Sigma-Aldrich, USA), anti-FSCN1 (Sigma-Aldrich, USA) and anti-VIME (Sigma-Aldrich, USA), anti-GST (Proteintech, USA), anti-Flag (MBL, USA). Detailed information of immunoblotting analysis was previously described42 .

Cells were lysed using RIPA buffer (Biosesang Inc, Seongnam, Korea) containing phosphatase and protease inhibitor cocktail (GeneDEPOT, Barker, TX, USA). Protein concentration was measured using Pierce™ 660 nm protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane (Merck, Kenilworth, NJ, USA). After blocking with 5% skim milk for 1 h, membranes were incubated overnight at 4 ℃ with primary antibodies, which were dissolved in PBST containing 5% BSA. The following primary antibodies were used: anti-Mindin (SPON2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)], anti-RBP-Jk (Santa Cruz Biotechnology), and anti-activated Notch1 (Abcam, Cambridge, UK), which detected Notch1 intracellular domain (N1ICD). Anti-GAPDH (Bioworld, Dublin, OH, USA) was used as control. Further, membranes were incubated with HRP-conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) for 90 min followed by detection using ECL kit (Bio-Rad, Hercules, CA, USA) and Supernova-Q1800.