Anti acetyl lysine

Approximately 1 mg of total proteins was incubated overnight at 4°C with anti‐Sirt1 antibody (Cell Signaling) or anti‐HIF‐1α antibody (Novus Biologicals, Littleton, CO, USA) followed by precipitation with 20 µl of protein A/G‐Plus‐Agarose (Santa Cruz, Dallas, TX, USA) for 4 hr at 4°C. The precipitated complexes were immunoblotted with anti‐Sirt1 (Cell Signaling), anti‐HIF‐1α (Novus Biologicals), or anti‐acetyl‐lysine (Cell Signaling).

S2 cells were lysed in ice-cold IP buffer (50 mM TRIS pH 8.0, 150 mM NaCl, 1% Triton-X and cOmplete ULTRA protease inhibitor, Roche) 48 hours after transfection for total protein lysate extraction. Cell lysates were centrifuged at 18000 rpm at 4 °C to collect the supernatant and total protein concentration was estimated by Bradford assay. Blots were probed with the primary antibodies anti-Cas9 (Abcam 191468, 1:500), anti-tubulin (Abcam 18251, 1:5000), anti-FLAG (Abcam 1162, 1:4000), anti-acetyl lysine (Cell Signaling Technology # 9441, 1:1000), and HRP coupled secondary anti-mouse (P0260) and anti-rabbit (P0448) antibodies (DAKO/Agilent, both at 1:2000). Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare) was used and signals imaged with a Chemi Doc XRS+ with Image Lab Software (BioRad).

Anti-HDAC6, anti-acetyl-α-tubulin, anti-acetyllysine, anti-Snail, and anti-GSK-3β antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-β-actin, anti-Hsp90, anti-ubiquitin, and protein A/G plus agarose were gained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-N-cadherin was purchased from Thermo Fisher Scientific Inc., (Taipei, Taiwan). Anti-vimentin and anti-E-cadherin antibodies were acquired from GeneTex, Inc. (Irvine, CA, USA) and BD Biosciences, Inc. (San Jose, CA, USA), respectively.

NPC cell lysates were prepared in IP buffer (1% Nonidet P^− 40, 150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 10 mM NaF, 1 mM Na₃VO₄, 10 mM N-ethyl-amide (NEM) and protease inhibitors [Complete protease inhibitor cocktail; Roche, Lewes, UK]). The pre-cleared lysate was immunoprecipitated with the indicated antibodies and with protein A/G-sepharose for 4 h. After that, the fractionated supernatant was transferred to the new tube. Samples and incubated overnight with 0.5 μg of primary antibody-FOXO3 (sc-11,351, Santa Cruz) [1 (link), 26 (link)], Anti-acetyl lysine (Cell-Signalling technology) or IgG negative control (2729, Cell-Signalling technology) (1500) at 4 °C on a rotator. The supernatant was discarded on the following day and beads were washed three times with PBS. Samples were boiled with 2 × SDS sample buffer for 5 min at 100 °C and were analysed by SDS-PAGE followed by western blotting.

Fixed tumor tissues were dehydrated, paraffin-embedded, and sectioned at 5 μm. After quenching endogenous peroxidase with 3% hydrogen peroxide for 15 min, sections were treated with normal horse serum. The sections were then incubated overnight at 4 °C with primary anti-proliferating cell nuclear antigen (PCNA) (PC 10; Sigma-Aldrich), anti-CD31 (polyclonal; Abcam, Cambridge, UK), anti-MMP-9 (D6O3H; Cell Signaling Technology) and anti-acetyl-lysine (polyclonal; Cell Signaling Technology) antibodies. HRP-conjugated anti-rabbit or anti-mouse IgG was next applied for 60 min at room temperature. Color was developed for 3 min by incubation with 3,3′-diaminobenzidine (DakoCytomation, Santa Clara, CA, USA). Sections were counterstained with hematoxylin and eosin (H&E) and examined under a Motic microscope (Motic, Richmond, BC, Canada) at 40× magnification.

INS-1 cells were treated with 3 μM A-485 for 6 h and harvested for endogenous immunoprecipitation. Protein lysates were incubated with anti-Hnf1α (Proteintech, Illinois, USA) at 4 °C for 2 h and then with Protein A/G PLUS-Agarose beads (Santa Cruz, Texas, USA) overnight at 4 °C. The binding complexes were harvested by centrifugation, washed with lysis buffer, and then eluted with loading buffer. Western blot was followed using anti-p300 (Santa Cruz) and anti-acetyllysine (Cell Signaling, Massachusetts, USA) antibodies for acetylation and binding detection.