Anti ub antibody

To analyze the polyubiquitination of ZFP36, we treated cells with 10 µM of the proteasome inhibitor MG132 for 6 h. The cells were washed with PBS, pelleted, and lysed in HEPES buffer (20 mM HEPES, pH 7.2, 50 mM NaCl, 1 mM NaF, 0.5% Triton X-100) plus 0.1% sodium dodecyl sulfate (SDS), 10 µM MG132, and protease-inhibitor cocktail. The lysates were centrifuged to obtain cytosolic proteins and incubated with anti-ZFP36 (Merck Millipore, ABE285) overnight. For denaturing conditions, cells were first denatured in lysis buffer containing 1% SDS in conjunction with heat inactivation at 100 °C for 10 min. The resulting sample is diluted tenfold in HEPES buffer to reduce the SDS concentration before the addition of an antibody for IP. The lysates were then pulled down with agarose beads. The beads were washed six times with HEPES buffer and then eluted with 200 μl of 1 M glycine (pH 3.0). The proteins were boiled in SDS-polyacrylamide gel electrophoresis sample buffer for 5 min and analyzed by immunoblotting with an anti-Ub antibody (Cell Signaling Technology, #3936).