Western lightening plus ecl kit

Individual cochlear epithelia were lyzed in RIPA lysis buffer (Sigma, no. R0278) supplemented with Roche Protease Inhibitor (Sigma, no. 11697498001) and Phosphatase Inhibitor Cocktail no.2 (Sigma, no. P5726) and no.3 (Sigma, no. P0044). Following manufacture recommendations, equal amounts of cochlear protein extract were resolved on NuPAGE 4–12% Bis-Tris Gels (Invitrogen, no. NP0322BOX) and transferred to Immun-Blot PVDF membrane (Bio-Rad) by electrophoresis. Membranes were blocked in 5% no-fat dry milk in TBST and immunoblotted with rabbit anti-P-Smad2/3 (1:1000; Cell Signaling, no.8828 RRID: AB_2631089), P-Smad1/5/9 (1:1000; Cell Signaling, no.13820, RRID: AB_2493181) and mouse anti-β-actin (1:1000; Santa Cruz, no. SC-47778, RRID: AB_626632). HRP-conjugated secondary antibodies from Jackson Immuno Research were used at a concentration of 1:10,000 (goat anti-rabbit IgG, no. 111-035-003; sheep anti-mouse IgG no. 515-035-003). Signal was revealed using a Western Lightening Plus ECL kit (Perkin Elmer, no. NEL120E001EA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, no. 34096) according to manufacturer’s instructions.

Cells were collected by centrifugation, washed in PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo 78442) with vortex vigorously. Lysates were then centrifuged at 11,000g at 4 °C for 10 min and the supernatant (quantify the concentration first) was transferred into 4× diluted Laemmli sample buffer containing 2-mercaptoethanol and boiled in 100 °C for 5 min. After that, lysates were separated on 10% Bis-Tris polyacrylamide gels and transferred to PVDF membrane (Millipore IPVH85R). The blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies. Blots were developed with the Western Lightening® Plus ECL kit (PerkinElmer). The primary antibodies including anti-Ppp2r1a (Genetex GTX-102206, 1:500), anti-phospho Tyr307 PP2AC (Santa Cruz sc-271903, 1:100), anti-Runx2 (Cell Signaling 8486, 1:1000), anti-human phospho Ser451 Runx2 (Bioss bs-5685, 1:300), anti-PPARγ (ABclonal A0270, 1:500), anti-phospho Ser492 BRD4 (Millipore ABE1451, 1:500), anti-Osterix (Bioss bs-1110, 1:300), anti-collagen X (Abcam ab58632, 1:100), anti-MMP13 (Genetex GTX-100665, 1:500) and anti-GAPDH (Genetex GTX-100118, 1:5000) were used. Immunoprecipitation was performed by Capturem™ IP & Co-IP Kit (Takara, 635721) and the results were analyzed with blotting the elution by anti- Ppp2r1a and anti- human phospho Ser451 Runx2.