Ab7090

Protein was extracted from tissues via RIPA (Beyotime, Beijing, China). The protein concentration was assessed by BCA kit (Beyotime). Then, protein samples were separated through SDS-PAGE, which was transferred onto PVDF membrane. The membrane was blocked by 0.5% skimmed milk for 2 h at room temperature, followed by incubation with primary antibodies against Protein Penk (1:1000; ab150346; Abcam, United States), C3 (1:1000; ab181147; Abcam), Fga (1:2000; ab108616; Abcam), Slc4a1 (1:1000; ab196798; Abcam), and β-actin (1:200; ab115777; Abcam) at 4°C overnight, and secondary antibodies (1:5000; ab7090) at room temperature for 2 h. Protein blots were analyzed through the Western Lighting Ultra (Thermo).

All protein was extracted from endplate chondrocytes using RIPA buffer (Aspen Biotechnology, Wuhan, China), and the protein concentration was measured using the BCA protein assay (Aspen). Then, the SDS-PAGE with a volume fraction of 10% was prepared and used to separate protein (40 μg/lane). Electrophoresis (100 V) was stopped when the target protein reached the middle of the gel. Next, protein was transferred onto PVDF membranes and blocked with TBST containing 5% skim milk for 1 h at room temperature. After that, the protein was incubated with primary antibodies (anti-cleaved caspase 3 (ab32042), anti-LC-3I/II (ab128025), anti-ATG7 (ab52472) and anti-β-Actin (ab8226)) overnight at 4°C. Subsequently, membranes were incubated with HRP-conjugated secondary antibody (ab7090, 1 : 5000) at room temperature for 1 h. Finally, an enhanced chemiluminescence (ECL) kit (Thermo) was used to observe protein bands. β-Actin is used as a loading internal control. All the antibodies were obtained from Abcam (Cambridge, MA, USA).