Versa doc molecular imager

Approximately, 7.5×10⁶ cells were resuspended in a 0.2ml extraction buffer (50mM Tris.HCl, pH6.8, 2% SDS, 10mM sodium fluoride) and sonicated with 4 cycles on a Sonics Vibra Cell machine. Sonicated extracts were centrifuged at 20,000xg for 10min at 4°C and supernatants were collected for protein estimation using DC^TM Protein Assay Reagent (Bio Rad, USA). 50μg protein was loaded in each well of 10% SDS-PAGE gel and transferred to PVDF membrane (Thermofisher). Blots were incubated in primary antibody diluted in 1xTBS, 5.0% BSA (Sigma, USA), 0.1% Tween-20. Primary antibodies against MYB (Milipore), BCL-2 (Santa Cruz Biotech), MCL-1 (Cell Signaling Technology, USA) were used at 1:000 dilution and those against β-actin (Santa Cruz Biotech), RNA PolII (anti phospho Ser2 and Ser5, Cell Signaling Technology, USA) were used at 1:500. Blots were developed using chemiluminescent reagent (Thermo Scientific) and the signals were detected with a Versa Doc Molecular Imager (Bio Rad).

Myosin was extracted from individual embryonic or adult mouse whole hearts as previously described70 (link) with minor modifications. Protein was quantified by Bradford Assay. Proteins were separated on 8–16% gradient Tris-glycine gels by electrophoresis and transferred to polyvinylidene difluoride membranes using standard western blotting procedures. Membranes were blocked with 5% bovine serum albumin, incubated with primary antibodies overnight, washed, incubated with secondary antibodies for 1 h, washed and imaged using a VersaDoc Molecular Imager (Bio-Rad). Antibody details are provided in Supplementary Table 11. Quantification of band intensities was carried out using the gel analyzer feature of ImageJ (ref. 71 (link)), with GAPDH serving as a loading control. Uncropped western blot images are provided in Supplementary Fig. 11.

The AF tissues from human IVDs with degenerative grades 2 to 4 (n = 3 IVDs per grade) were incubated in guanidine hydrochloride buffer (4 M guanidinium chloride, 50 mM sodium acetate, and 10 mM ethylenediaminetetraacetic acid) for 72 hours. NGF content was determined by Western blotting of extracts. Briefly, extracts were electrophoresed on 4%‐20% gradient gels (Bio‐Rad, Hercules, CA) and transferred to polyvinylidene fluoride (PVDF) membrane. Blots were probed with anti‐NGF antibody. Blots were developed by incubation with antirabbit immunoglobulin G antibody conjugated with horseradish peroxidase and Amersham ECL Prime chemiluminescent detection reagent (GE Healthcare, Piscataway, NJ). Images were captured on a VersaDoc molecular imager (Bio‐Rad, Hercules, CA).