Super ecl chemiluminescence solution

Total protein from mice skin tissues or melanocytes was extracted using total protein extraction reagent (Beyotime, Shanghai, China) according to the manufacturer’s instructions, and the concentration was measured using a nucleic acid/protein analyzer. Two hundred micrograms of protein lysate from each sample were size-separated by SDS-PAGE electrophoresis and transferred onto nitrocellulose filter membranes (Boster, Wuhan, China). The membranes were blocked in 5% skimmed milk powder (Boster, Wuhan, China) at room temperature for 1 h and then were probed with the primary antibody diluted in Tris-buffered saline-Tween (TBST) overnight at 4 °C. The next day, the membranes were washed 3 times in TBST for 10 min each and incubated with HRP-conjugated second antibody (1:10,000 (v/v), Boster, Wuhan, China) at 37 °C with horizontal shaking for 1 h. Subsequently, the membranes were washed 6 times in TBST for 5 min each, and the proteins were visualized via a super ECL chemiluminescence solution (Boster, Wuhan, China). The Western blot results were analyzed using Image-ProPlus 6.0 software (Olympus, Hatayaga, Japan) to measure the area and gray value for each target band. The target protein expression level was normalized relative to the corresponding internal reference level in each lane.

Protein concentrations in the supernatants were measured using a BCA protein assay kit (Boster). Then, equal amounts of protein lysate were loaded on a 10% SDS‐polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% BSA at 37°C for 60 minutes, and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used in this study: MMP1 (#10371‐2‐AP; 1:1500), MMP3 (#17873‐1‐AP; 1:1000), HMGB1 (#10829‐1‐AP; 1:1000), TLR4 (#19811‐1‐AP; 1:1000) and PI3K (#20584‐1‐AP; 1:1000) from Proteintech Group; Collagen2 (#ab34712; 1:1000), MMP13 (#ab39012; 1:1000), iNOS (#ab3523; 1:500), Aggrecan (#ab3778; 1:1000) from Abcam (Cambridge, MA); CCN3 (#8767; 1:1000), P‐AKT (#4060; 1:1000), AKT (#4685; 1:1000), P‐mTOR (#5536; 1:1000), mTOR (#2983; 1:1000), P‐PI3K (#4228; 1:1000), Atg5 (#12994; 1:1000), Beclin1 (#3495; 1:1000) and LC3‐I/II (#12741; 1:1000) from Cell Signalling Technology; ADAMTS5 (#BA3020; 1:500) and GAPDH (#BM3876; 1:500) from Boster. Next, the blots were incubated with goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Boster, 1;5000) for 1 hour at 37°C. Protein bands were then detected using a super ECL chemiluminescence solution (Boster). The protein expression was normalized to GAPDH and quantified using ImageJ software.