Anti gcn5

20μg of protein was electrophoresed on 4-12% Nu-Page gel (Life Technologies) and transferred to a nitrocellulose membrane. Membranes were blocked in 5% milk in TBST at room temperature for 30 minutes. Membranes were incubated overnight at 4 degrees Celsius with the primary antibodies: anti-GCN5 (1:1000), anti-MYC (1:1000), anti-AKT (1:1000), anti-phospho-AKT (1:1000), anti-SYK (1:1000), anti-BTK (1:1000), anti-FOXO1 (1:1000), anti-phospho-FOXO1 (1:1000), and anti-PARP (1:1000) (Cell Signaling); anti-Ada2b (1:500); anti-Beta Actin (1:5000) (Santa Cruz), anti-USP22 (1:1000) (homemade); H3 acetyl lysine 9 (1:500) (Millipore); H3(1:10,000) (Abcam). Membranes were incubated in secondary-horseradish peroxidase (HRP) conjugated antibodies (GE Healthcare, Cat. # NA934V for Rabbit, NA931V for Mouse) for 1 hour at room temperature. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for chemiluminescent protein detection.

Cell pellets were dried, and then cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The following antibodies were used for detection of the desired proteins: PCAF (1:1000, sc13124, Santa Cruz), anti-GCN5 (1:1000, #3305, Cell Signalling), anti-PML (1:1000, sc966, Santa Cruz), α-tubulin (1:1000, T6199, Sigma-Aldrich), and β-actin (1:1000, A1978, Sigma-Aldrich).

Cells were washed in phosphate‐buffered saline (PBS, Thermo Fisher Scientific) and lysed using protein extraction buffer (10 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1% sodium deoxycholate (NaDoc), 0.1% sodium dodecyl sulphate (SDS) and 1% glycerol) supplemented with protease and phosphatase inhibitor mix (Roche). Total lysates were quantified using BCA Protein Assay Kit (Life Technologies). 15 µg of proteins was separated by SDS‐PAGE on precast gradient (4–12%) gels (Invitrogen) and transferred onto nitrocellulose membrane in Transfer buffer (Invitrogen) supplemented with 10% (vol/vol) methanol. Membranes were blocked in PBS 0.05% Tween 5% non‐fat dry milk for 1 h at room temperature (RT) and then incubated overnight at 4°C with anti‐GCN5 (1:1000, Cell Signaling, #3305, rabbit) and anti‐GAPDH (1:5000, Santa Cruz, Sc‐32233, mouse). After washing, membranes were incubated with the appropriate HRP‐conjugated secondary antibody for 1 h at RT. Detection was performed using the enhanced chemiluminescence system (ECL, Pierce™ ECL Western Blotting Substrate kit). Images were acquired with the ChemiDoc MP Imaging System (Bio‐rad) and quantified using Image Lab software 5.2.1 (Bio‐Rad).