Ab34712

For further analysis of chondrogenic markers, the NSCs were lysed using RIPA lysis buffer (Sigma-Aldrich). A total of 30 µg protein was subject to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with the use of 10 % resolving gels, then followed by transfer to polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with primary antibodies specific for COL II (Abcam, ab34712), SOX9 (Santa Cruz, sc-166,505), Aggrecan (Santa Cruz, sc-70,332), COL I (Abcam, ab34710), and β-actin (Santa Cruz, sc-47,778), respectively, all of which were diluted in 1:1000. Those markers were visualized by chemiluminescence using Western ECL substrate (Thermo Scientific), and the luminescent images were analyzed using a LAS-3000 (Fujifilm).

After culture, the discs were rinsed with PBS and the innermost NP tissue was isolated under a dissecting microscope. Then, Western blotting assay for SOX-9, aggrecan, and collagen II was performed. Briefly, after the total protein was extracted using RIPA lysis buffer (Beyotime, China), equal protein sample in each group was subjected to SDS/PAGE system and transferred to the PVDF membrane. Then, incubation of the primary antibodies (GAPDH: Abcam, ab8245; collagen II: Abcam, ab34712; aggrecan: Santa Cruz, sc-16492; SOX9: Santa Cruz, sc-20095; all were diluted 1:1000) was performed at 4°C overnight, and incubation of the corresponding HRP-conjugated secondary antibodies (goat anti-mouse IgG, goat anti-rabbit IgG, and mouse anti-goat IgG, ZSGB-BIO, China, diluted 1:1000) was performed at room temperature for 2 h. Thereafter, protein bands on the PVDF membrane were visualized using ECL Plus reagent (Thermo, U.S.A.). Then, protein expression normalized to GAPDH was calculated according to the gray value that was measured using the ImageJ software (National Institutes of Health, U.S.A.).

Briefly, total protein was extracted using the RIPA lysis buffer (Beyotime, China) and subjected to the SDS/PAGE system (6% separate gel). After the protein sample was transferred on to the PVDF membranes, the PVDF membranes were sequentially incubated with primary antibodies (GAPDH: Abcam, ab8245; collagen II: Abcam, ab34712; aggrecan: Santa Cruz Biotechnology, sc-16492; SOX9: Abcam, ab185966. All primary antibodies were diluted at 1:1000) at 4°C overnight, and the corresponding HRP-conjugated secondary antibodies (goat anti-mouse IgG, goat anti-rabbit IgG, and mouse anti-goat IgG, ZSGB-BIO, China, diluted at 1:1000) at room temperature for 2 h. Finally, ECL Plus reagent (Thermo, U.S.A.) was used to develop the protein bands on PVDF membranes. After the gray value of protein bands was calculated using the ImageJ software (National Institutes of Health, U.S.A.), it was normalized to that of GAPDH.