Col 3

RIPA lysis buffer (Cat: C1053, Applygen, China) containing Cocktail (50x) (Cat: P1265, Applygen, China) was used to prepare tissues and cells. After isolating proteins from homogenates, immunoblotting was performed overnight at 4°C using the following rabbit primary antibodies: Col III (Cat: 22734-1-AP, dilution: 1 : 1000, Proteintech, USA), Col I (Cat: 66761-1-Ig, dilution: 1 : 1000, Proteintech, USA), CD9 (Cat: ab263019, dilution: 1 : 1000,Abcam, UK), CD63 (Cat: ab134045, dilution: 1 : 1000, Abcam, UK), TSG101 (Cat: ab133586, dilution: 1 : 1000, Abcam, UK), TNC (Cat: 67710-1-Ig, dilution: 1 : 1000, Proteintech, USA), TNMD (Cat: ab203676, dilution: 1 : 1000, Abcam, UK), SCXA (Cat: DF13293, dilution: 1 : 1000, Affinity, USA), PCNA (Cat: 10205-2-AP, dilution: 1 : 1000, Proteintech, USA), poly ADP-ribose polymerase 1 (PARP1, Cat: 13371-1-AP, dilution: 1 : 1000, Proteintech, USA), catalase (Cat: 21260-1-AP, dilution: 1 : 1000, Proteintech, USA), and GAPDH (Cat: 60004-1-Ig, dilution: 1 : 1000, Proteintech, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies (Cat: 15015&15014, dilution: 1 : 10000, Proteintech, China) were then incubated with the membranes for 1 h at room temperature. Chemiluminescent signals were developed with an enhanced chemiluminescence (Millipore, USA) and detected by the ChemiDoc imaging system (Tanon, China).

The cells are cleaved, and proteins were extracted from the supernatant. The protein was quantified by a BCA Protein Assay Kit (Beyotime). Then, 50 μg of total cell protein dissolution products was isolated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) membranes (PALL). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was diluted at 1:4,000; PRDX2 (1:1,000; Abcam, USA and Proteintech, China), COL I (1:1,000; Abcam, USA), COL III (1:1,000; Proteintech, China), VCAM-1 (1:1,000; Abcam, USA), ICAM-1 (1:1,000; Abcam, USA), p-p38 (1:1,000; Abcam, USA), p38 (1:1,000; Abcam, USA), p-JNK (1:1,000; Abcam, USA), JNK (1:1,000; Abcam, USA), p-ERK (1:2,000; Proteintech, China), and ERK (1:1,000; Proteintech, China) antibodies were incubated at 4°C overnight after washing three times with Tris-buffered saline–Tween-20 (TBST). The membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000; GE, HyClone) at 37°C for 1 h. The protein bands were visualized by enhanced chemiluminescence (ECL, Advansta) using a ChemiDoc™ MP imaging system (BIO-RAD).