Semi-Native Gel Electrophoresis

Semi-native gel electrophoresis was adapted from previously described for verification of NPM oligomerization (Hamilton et al., 2014). Cells were exposed to UV radiation (or not, control non-irradiated) and after 3 h were washed thrice with cold PBS and lysed on ice for 10 min in cold lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na₃V0₄, 10 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, proteases, and phosphatases inhibitors used as described). The lysates were rotated for 25 min at 4°C and centrifuged (21,000 × g, 15 min, 4°C). Quantification was done immediately, and 400 μg of a total protein was diluted in a sample buffer (62.5 mM Tris–HCl, 10% glycerol, 0.01% bromophenol blue) without boiling. Samples were loaded onto 10% Bis-Tris medium gel (1/3 Bis-Tris 1 M, acrylamide to 10% for resolving and 5% for stacking gel, plus APS and Temed). Gels were run (250 mM MOPS, 250 mM Tris, 5 mM EDTA, 0.5% SDS) at a constant voltage (50 V) at 4°C overnight and transferred to a nitrocellulose membrane and incubated as previously detailed for immunoblottings.