Beyoecl plus chemical luminescence solution

Western blot analysis for BCL2 and CCND1 levels was performed as described by Li et al. [30 (link)], with minor modifications. Briefly, cells were lysed, and the protein concentration of total cell lysates was measured by using the BCA assay (Beyotime, China). About 25 μg of whole cell lysates were subjected to electrophoresis and then transferred to an Immobilon-P Membrane (Merck Millipore, USA). After blocked by 5% non-fat milk for 1 hour at room temperature, the membranes were incubated with rabbit anti-human BCL2 or rabbit anti-human CCND1 (both 1: 1000, Cell Signaling, USA) and mouse anti-human β-actin (1: 2000, Abcam, USA) for overnight at 4°C. After incubation with corresponding secondary antibodies for 1 hour, the membrane was washed 3 times with TBST, and then incubated with BeyoECL Plus chemical luminescence solution (Beyotime, China). The membrane was then imaged using a ChemiDoc XRS imaging system and analyzed using the QuantityOne software (Bio-Rad, USA).

Cells were lysed with radioimmunoprecipitation assay lysis buffer (1% NP-40, 0.5% Sodium deoxycholate and 0.1% SDS; Wuhan Boster Biological Technology, Ltd.) and protein concentration of whole cell lysates was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Briefly, 30 µg whole cell lysates were loaded and separated by electrophoresis on 10% SDS-polyacrylamide gels and were then transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with rabbit anti-human TIMP3 (cat no. ab39184; 1:1,000; Abcam, Cambridge, MA, USA) and rabbit anti-human β-actin (cat no. ab8227; 1:2,000; Abcam) overnight at 4°C. The membranes were then washed three times with TBST containing 0.1% Tween-20 and incubated with goat anti-rabbit secondary antibody (cat no. A0208; 1:1,000; Beyotime Institute of Biotechnology) for 1 h at room temperature. Subsequently, membranes were washed a further three times with TBST and were incubated with BeyoECL Plus chemical luminescence solution (Beyotime Institute of Biotechnology). The protein bands were visualized using a ChemiDoc XRS imaging system and were analyzed using Quantity One software version 4.3.0 (Bio-Rad Laboratories, Inc.).