Bicinchoninic assay kit

Total protein of PC12 cells in each group was extracted with a protein extraction kit (Beyotime). Protein concentrations were detected with a bicinchoninic assay kit (Thermo Scientific). Forty micrograms of protein were loaded and then separated on sodium dodecyl sulfate–polyacrylamide gels (10%). Next, target proteins were electronically transferred to polyvinylidene difluoride membranes using semi-dry transfer at 25 V for 7 minutes. Subsequently, membranes were blocked with 5% skim milk powder at room temperature for 2 hours, followed by primary antibodies [rabbit polyclonal anti-ChAT (1:800; Abcam) and mouse monoclonal anti-β-actin (1:2000; Beyotime)] at 4°C overnight. Secondary horseradish peroxidase-conjugated anti-rabbit IgG (1:1000; Bioss, Woburn, MA, USA) and anti-mouse IgG (1:1000; Bioss) were used to detect related proteins for 2 hours at room temperature. Finally, Enhanced Chemiluminescence-Plus reagent (Bio-Rad, Hercules, CA, USA) and Software Quantity One (Bio-Rad) were used to visualize immunoblots, and relative protein expression was calculated with β-actin as the internal reference.

Cells were resuspended in lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min and sonicated for 2 min at 20 W, followed by centrifugation at 12,000 × g for 10 min at 4°C. The supernatant was collected and 50 µg/lane protein (concentration determined using the bicinchoninic assay kit (Thermo Fisher Scientific, Inc.) was separated using SDS-PAGE on a 10% gel (Bio-Rad Laboratories, Inc.) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.), which was blocked in Tris-buffered saline/Tween-20 (TBST) with 5% non-fat milk for 1 h at 37°C. The membrane was subsequently incubated with primary antibodies against TGF-β1 (cat no. sc-146; 1:2,000), PKCβI (cat no. sc- 209; 1:1,000) and GAPDH (cat no. sc-25778; 1:500) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated labeled goat anti-rabbit secondary antibody (cat no. sc-2004; 1:500; Santa Cruz Biotechnology, Inc.) for 1 h at 4°C, followed by additional three washes with TBST. Protein bands were visualized by enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA). The Scion Image system version 4.03 (National Institutes of Health, Bethesda, MD, USA) was used to quantify band intensity and data are expressed as the mean of three independent experiments.

The protein was extracted from 10 mg articular cartilage using a protein isolation kit (ReadyPrep; GE Healthcare Life Sciences). The protein concentration was determined using a bicinchoninic assay kit (Thermo Fisher Scientific, Inc.). The protein (20 μg) was separated on 12% SDS PAGE gel and transferred to the nitrocellulose membrane. At room temperature, the membrane was sealed in 5% skimmed milk for 2 hours and incubated with the following primary antibodies at 4°C overnight: Anti-LC3 I, Anti-LC3 II, anti-MMP13, anti-aggrecan, anti-collagen II, anti-TGFβR1, anti-β-actin (Cell Signaling Technology, Inc., Danvers, USA). The next day, the nitrocellulose membranes were washed three times and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The protein bands were detected by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) scanned by ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein expression was normalized to β-actin and densitometric analysis was performed by ImageJ Software version 7.0 (National Institutes of Health, Bethesda, MD, USA).