Anti ccne2

6-well plates were used for culturing HCFs. HCFs were collected after treatments. BCA protein assay kit was used for determining the concentrations of cell lysates. 1 mg of total proteins were used for immunoprecipitation. The samples were incubated in rabbit-controlled Ig-G antibody and anti-E2F1 (1:100, Proteintech, Wuhan, China) antibody under condition of 4°C overnight. Then the samples were suspended with 25 μL of protein A/G PLUS-Agarose. After 2 h of incubation and washing, the samples were western-blotted, and anti-CCNE2 (1:1000, Abclonal, Wuhan, China) antibody was incubated to evaluate the conjunction amount of E2F1 and CCNE2.

RIPA (Beyotime, China) was used for harvesting cell lysates, and BCA protein assay kit (Beyotime, USA) was used for determining the concentration of protein samples. Same amounts of proteins were separated by SDS-PAGE gels (10%) and transferred to PVDF membranes. After 2 h of blocking in 5% Bovine Serum Albumin (BSA, Thermofisher, USA), PVDF membranes were incubated in primary antibodies under the temperature of room (24°C) for 2 h. After that, PVDF membranes were washed 3 times in 15 min with TBST and incubated in horseradish peroxidase-conjugated secondary antibody (Beyotime, China) for 1 hunder 24°C. Finally, an enhanced chemiluminescence (ECL, Vazyme, Nanjing, China) was used for imaging western blots. Image data were analyzed by ImageJ software (NIH, MD, USA). The antibodies used in this experiment are listed as below Anti-FN-1 (Rabbit, 1:3000, Abcam, MA, USA), anti-Col-1a1 (Rabbit, 1:1000, Abclonal, Wuhan, China), anti-α-SMA (Rabbit, 1:1000, Abcam, MA, USA), anti-E2F1 (Rabbit, 1:1000, Abclonal, Wuhan, China), anti-CCNE2 (Rabbit, 1:1000, Abclonal, Wuhan, China), anti-GAPDH (Rabbit, 1:3000, CST, MA, USA) for western-blot.