Anti gapdh polyclonal antibody

Aliquots of 20 µg of total protein extracts were size separated in a 4–12% (w/v) gradient polyacrylamide precast gel containing 0.1% (w/v) SDS (NuPage, Invitrogen, Carlsbad, CA, USA) and transferred onto a nitrocellulose membrane (Biorad, Richmond, CA, USA). Detection of pIKBα and GAPDH was performed through overnight (O/N) incubation with the pIKBα pSer32 ABfinity Rabbit Monoclonal antibody (2.5 μg/µL, Thermo Fisher, Waltham, MA, USA) and anti-GAPDH polyclonal antibody (1:500, Santa Cruz Biotechnology). Detection was achieved using Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody and Western Lightning Plus-ECL (PerkinElmer Inc., Waltham, MA, USA). Image acquisition was obtained using an IQ LAS 4000 mini biomolecular imager.

Cells were lysed with phospho-protein extraction buffer (Merck Millipore) supplemented with protease-phosphatase cocktail inhibitor (Sigma). 40 μg total lysates were then resolved on a 10% or 15% Tris-HCl gel and immunoblotted with the following specific antibodies: anti-BAX (6A7) monoclonal antibody (Santa Cruz Biotechnology, Dallas TX), anti-p21 (H-164) polyclonal antibody (Santa Cruz Biotechnology), anti-PARP polyclonal antibody (Cell Signaling Technologies), anti-BCL2 (clone100) monoclonal antibody (Merck Millipore), anti-caspase 3 (8G10) monoclonal antibody (Cell Signaling Technologies), anti-GAPDH polyclonal antibody (Santa Cruz Biotechnology).

Total protein was extracted, and the protein concentration was quantified using a BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). A total of 20 μg of protein from each sample was used for Western blotting. The samples were separated by SDS-PAGE(10%) at 200 V for 50 min. After transferring the proteins onto polyvinylidene fluoride (PVDF) membranes, the blotting was performed at 300 mA for 45 min. After blocking with 5% (w/v) dry milk in TBS for 1 h at room temperature. Membranes were incubated with the primary antibodies. The primary antibodies used in this study included monoclonal anti-Collagen II (1:1000), Aggrecan (1:500) (Cell Signaling Technology, Beverly, MA), Fos (1:500), p38 (1:500), JNK (1:500) and their phosphorylated antibodies (1:500, Santa Cruz, CA, USA) and anti-GAPDH polyclonal antibody (1:2000 Santa Cruz, CA, USA) at 4 °C overnight. Then the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibody (1:10000, Cell Signaling Technology, Beverly, MA) for 2 h at room temperature. Finally, the blots were developed with an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA), and the bands were quantified densitometrically using a Bio-Rad imaging system (Hercules, CA). GAPDH was used as the loading control.