Histone h4 antibody

Commercially available Curcumin (#81025.1), Anacardic acid (#13144), Garcinol (#10566) and IsoGarcinol (#21164) were purchased from Cayman. Anti-Flag affinity gel (#A2220) and anti-Flag antibody conjugated with horseradish peroxidase (#A8592) were purchased from Sigma-Aldrich. Acetyl-Histone H3 Antibody (#9675) and acetyl histone H4 antibody (#13534) were purchased from Cell Signaling Technology. SHMT2 (mSHMT Antibody (F-11): sc-390641- Non-conjugated), ß-actin antibody (SC-47778 HRP) and goat anti-mouse IgG-HRP (sc-2005) were purchased from Santa Cruz. And histone H4 antibody (#ab197517) was purchased from Abcam. Triple Flag peptide for eluting Flag-tagged HDAC11 enzyme and protease inhibitor cocktail were purchased from Sigma-Aldrich. ECL plus western blotting detection reagent and universal nuclease for cell lysis were purchased from Thermo Scientific Pierce. Acyl peptides (myristoyl-H3K9) and Alk14 were synthesized.^{26 (link)} The peptide sequence for the H3K9 peptide is KQTARK(myristoyl)STGGWW with uncapped N-terminal and SHMT2 peptide is SDEVK(myristoyl)AHLLAWW with capped acetyl N-terminal.

HeLa cells were cultured in 10-cm dishes. 80~90% confluent HeLa cells were treated with the corresponding inhibitors (DMSO, 0.1 μM of TSA, 0.1 μM of TSA + 70 μM of CPTH6, 0.1 μM of TSA + 20 μM of Garcinol, and 0.1 μM of TSA + 40 μM of Garcinol) for 6 h. After 6 h, cell culture media were removed and 5 ml of PBS was added into each dish. The cells were scraped off the plates and collected by centrifugation at 500 g for 5 min. Collected cells were washed twice with 1 ml of PBS. After removing PBS gently removed by pipetting, cells were then lysed in 50 μL of 4% SDS lysis buffer (50 mM triethanolamine at pH 7.4, 150 mM NaCl, 4% (w/v) SDS) with protease inhibitor cocktail (1:100 dilution) and nuclease (1:1000 dilution). After vortexing, the samples were incubated at room temperature for 15 min. The protein concentration in the total cell lysate was determined using a BCA assay and an equal amount of protein was loaded onto a 15% SDS-PAGE gel and resolved. The gel was used for western blot analysis with acetyl histone H4 antibody (Cell Signaling, #13534) and histone H4 antibody (Abcam, #ab197517).