Anti acetyl α tubulin antibody

HeLa cells that were incubated with SirReal2 (20 and 50 μM), SirReal6 (50 μM), AGK2 (Sigma-Aldrich, 20 μM) or DMSO as a control in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, antibiotics and DMSO (1% (v/v)) for 4 h, were fixed with ice-cold methanol (10 min), washed with PBS and blocked with PBS supplemented with 0.1% (v/v) Triton-X-100 and 5% (v/v) FCS (30 min). Cells were then stained with an anti-acetyl-α-tubulin antibody (Sigma-Aldrich, T6793) and then probed with a secondary Alexa 546 conjugated anti-mouse-antibody (Invitrogen). Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Coverslips were mounted with FluoroMount (Sigma-Aldrich) and sealed with DPX Mountant (Sigma-Aldrich). Images of the mounted samples were acquired on a Leica DM500 microscope equipped with a Leica DFC 395 FX camera and HBO 100 W lamp40 (link). The microscope was run with the Leica Application Suite 4.4.0 software. Chroma UV filter set (No. C40888) and Leica N2.1 filter set (No. 513832) were used for DAPI and Alexa 546 signal acquisition, respectively, with a HCX FL Fluotar 40x/0.75 (dry) objective. Further details about the equipment and the settings that were used to acquire the images are found in the Supplementary Methods section. Unprocessed images are found in Supplementary Fig. 9.

HL60 cells were treated with the inhibitors in the given concentrations. Cells treated with dimethyl sulfoxide as a vehicle were used as controls. After an incubation time of 4 h, cells were transferred into 50-mL Falcon tubes and centrifuged (300× g, 5 min). The supernatant was removed, and the cells were washed with PBS, before they were transferred to 1.7-mL reaction tubes. The cells were lysated with Bio-Rad loading buffer, and the cell suspension was heated to 95 °C for 5 min. The proteins then were separated using a 15% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Roti-PVDF, Carl Roth, Karlsruhe, Germany). Acetylation levels were detected using an anti-acetyl histone H3 antibody (Millipore, Billerica, MA, USA) and an anti-acetyl-α-tubulin antibody (Sigma Aldrich, St. Louis, MO, USA). An antibody directed against GAPDH (Sigma Aldrich) was used as a loading control.

HeLa cells were plated in petri dishes (5 cm, PAA), incubated overnight to a confluency of 30–40% and then treated with SirReal2 dissolved in RPMI1640 medium supplemented with fresh 20% (v/v) FCS (PAA), 1% (v/v) penicillin (PAA), 1% (v/v) streptomycin (PAA), 1% (v/v), L-glutamine (PAA), 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 (Sigma-Aldrich), 2% (v/v) IGEPAL (Sigma-Aldrich), 2% (w/v) SDS, 10% (v/v) glycerol, 50 mM NCA (Sigma-Aldrich), 3.3 μM trichostatin A (Sigma-Aldrich), 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, Sigma-Aldrich, T6793) and an anti-GAPDH antibody (1:2,000–1:5,000, Sigma-Aldrich, G9545) as a loading control (Fusion SL, peqlab). An uncropped blot is shown in Supplementary Fig. 8.