Acetylated lysine antibody

EIF2α, p-EIF2α (Ser51), RelA, NFATc1, c-fos, ATF3, HSF1, p-HSF1 (Ser320), p24, Lamin A/C, β-actin antibodies and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Acetylated lysine antibody, p300, CDK9 and Cyclin T1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human FITC conjugated anti-CD25 and PE conjugated anti-CD69 antibodies were purchased from BD Biosciences (San Jose, CA, USA). PHA, poly I:C, STA-4783, thapsigargin, prostratin, PMA, ionomycin, SAHA, JQ1, dilazep, TNF-α, hemin, salubrinal, MG-132, BAY 11-7082, CsA, C646 and EX527 were from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol, parthenolide (PTN), Ver-155008, KRIBB11, 17-DMAG and NVP-AUY922 were from Merck Calbiochem (Darmstadt, Germany). PEZ-HSF1 was purchased from ViGene Bioscience Inc (MD, USA). NL4-3E-R-luc plasmid, J-Lat 10.613 (link), U141 (link) and ACH242 (link) cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO₂. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.

For Western blotting, transfected cells were harvested and lysed in lysis buffer (50 mM Tris-Cl (pH-8.0), 150 mM NaCl, 1 mM DTT, 5 mM EDTA, 0.5% NP-40 and 1X protease inhibitor cocktail (Amresco) followed by sonication. Protein samples were resolved by SDS-PAGE, transferred onto the methanol-charged PVDF membrane using semi-dry transfer apparatus. Subsequently, the blots were probed with indicated primary antibodies. RFP antibody, GFP antibody, GST antibody, PXR antibody, His antibody and acetylated-lysine antibody were purchased from Santa Cruz Biotechnologies. TIP60 antibody, RXRα antibody and β-actin antibody were procured from Cell Signaling Technology (CST). Acetylated Histone H4 and Histone antibodies were purchased from Millipore. Horseradish peroxidase-conjugated (HRP) anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnologies while light chain specific HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Millipore. Blots were developed using ECL reagent (BioRad) and C digit scanner (Licor).