| Cell experiments |
HeLa cells were plated in petri dishes (5?cm), incubated overnight to a confluency of 30–40% and then treated with SirReal2 dissolved in RPMI1640 medium supplemented with fresh 20% (v/v) FCS, 1% (v/v) penicillin, 1% (v/v) streptomycin, 1% (v/v), L-glutamine, 1% (v/v) DMSO for 5?h at various concentrations. Cells were then washed with prewarmed PBS (2?ml), lysed in SDS–PAGE sample buffer (70?μl, 50?mM Tris/HCl, 0.5?mM EDTA, 1 × Complete Protease Inhibitors, 2% (v/v) IGEPAL, 2% (w/v) SDS, 10% (v/v) glycerol, 50?mM NCA, 3.3?μM trichostatin A, 50?mM DTT, 0.01% (w/v) bromophenol blue, pH 6.8) and sonicated (5?min). Cell samples were then separated using SDS–PAGE (12.5% (w/v) polyacrylamide), transferred to an activated nitrocellulose membrane (Bio-Rad), blocked with non-fat dry milk (Roth, 5% (w/v), TBS, 0.1% (v/v) Tween 20) and probed with an anti-acetyl-α-tubulin antibody (1:1,000) and an anti-GAPDH antibody (1:2,000–1:5,000) as a loading control. |
