PAR assay

Previously dissected and flash-frozen in LN2 stored long term at −80°C mouse quadriceps were pulverized with a frozen metal motar and pestle on dry ice. Pulverized quadricep samples were lysed in RIPA buffer (50 mM Tris, pH 7.5, 0.4M NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT), plus 50 μg/mL PMSF, 10 μM tannic acid and one protease inhibitor tablet (Thermo Scientific, A32963) per 10 mL of lysis buffer and prepared as outlined above. Lysates were pre-cleared on a rotator overnight at 4°C with Negative Control Magnetic Resin (Tulip Biolabs, #2427). The next day, the beads were removed and PAR WWE Domain Magnetic Affinity Resin assay (Tulip Biolabs, #2438) beads were added to the samples. Samples were incubated overnight at 4°C on a rotator. The lysates were removed, the resin was washed with the lysis buffer 4 times and once with PBS, laemmli buffer was added, boiled at 95°C for 1 min, and samples were removed from the resin.