Bicinchoninic acid bca method

Passage 2-4 chondrocytes were used for protein extraction. Briefly,
chondrocytes were washed with PBS 3 times and lysed with
radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China)
supplemented with 1 mM protease inhibitor cocktail and 1 mM phosphatase
inhibitor cocktail (Thermo, USA). The mixture was homogenized and lysed on
ice and centrifuged at 12,000g for 30 minutes at 4 °C. The
resulting supernatant was collected. Protein concentration was determined by
the bicinchoninic acid (BCA) method (Beyotime). The same amount of protein
sample was electrophoresed and transferred to the polyvinylidene fluoride
(PVDF) membrane (Millipore, USA). The membrane was blocked with 5% skim milk
for 1 hour at room temperature and incubated at 4 °C overnight with the
primary antibody against SOX9 (Abcam), Casp1 (Abcam), IL-1β (Abcam), and
β-actin (Abcam). After washing, the membrane was incubated with secondary
antibodies (Abcam) at room temperature for 1 hour. The protein bands were
exposed using ChemiDoc MP (Bio-Rad). Integrated density for protein bands
was determined using Image J (National Institutes of Health, USA).

PC-3 cells were seeded in 6-well or 12-well plates and left untreated (control) or treated with carbachol (2 μg ml⁻¹) or pirenzepine (110 μg ml⁻¹) for 2, 4, 6, 12, and 24 h. Cells were lysed in NP 40 buffer (Solarbio, Beijing, China) containing protease inhibitors. Total protein was measured using the Bicinchoninic acid (BCA) method (Beyotime). Primary antibodies against SHH (1:2000, Abcam, Cambridge, MA, USA), PTCH1 (1:1000, Abcam), GLI1 (1:1000, CST, Beverly, MA, USA), and CHRM1 (1:1000, Abcam) as well as horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSbio, Beijing, China) were applied. Equal protein sample loading was monitored using an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000, Ray Antibody, Beijing, China). The probed proteins were visualized with an enhanced chemiluminescence (ECL) blotting detection kit (BIO-RAD, Chengdu, China).