Acetyl histone h3 lys9 lys14

Total protein from the cells was precipitated by the addition of 6% TCA, and equal amounts of protein were loaded on 10% acrylamide gels. Samples were analyzed by Western blot as described previously (10 (link)). The following primary antibodies were applied: mouse monoclonal anti-PMCA4b (JA3, recognizing the region between residues 1,156–1,180, which is specific to hPMCA4b (26 (link)), dilution 1:1,000), anti-pan PMCA (5F10, dilution 1:5,000) (27 (link)), rabbit polyclonal anti-PMCA1 (Affinity BioReagents, PA1-914, dilution 1:1,000), mouse monoclonal anti-SERCA2 (IID8, dilution 1:2,500, Sigma-Aldrich, S1439), mouse monoclonal anti-SERCA3 [PL/IM430, dilution 1:200 (10 (link))], rabbit monoclonal anti-phospho-p44/42MAPK (ERK1/2) (cell Signaling, CST4370S, dilution 1:1,000), mouse monoclonal anti-ERK1/2 (MK1) (Santa Cruz, sc135900, dilution 1:500), rabbit polyclonal anti-beta-tubulin (Abcam, ab6046), rabbit polyclonal acetyl-Histone H3 (Lys 9/Lys 14) (cell Signaling, 9677). Subsequently HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch, dilution 1:10,000) were used, and detection was performed with Pierce ECL Western Blotting Substrate (Thermo Scientific) and luminography. Densitometric analysis was performed by ImageJ software v1.42q.

In sterile 6‐well plates, Day 8 BMMØs were seeded at 1 × 10⁶ cells/well and incubated at 37°C and 5% CO₂ overnight to allow for cell adherence, as previously described.
¹⁷ (link) BMMØs were treated with 300 μg/mL iNP, 300 μg/mL iNP‐SAHA_Low, or 300 μg/mL iNP‐SAHA_High and incubated for 4, 9, 27, or 48 h. Cells were isolated using RIPA Buffer (#R0278) (Millipore Sigma, St. Louis, MO) containing Halt™ Protease Inhibitor Cocktail (#78429) (Thermo Fisher, Waltham MA) and scraped using a cell scraper (VWR, Radnor, PA). A 1/8″ tip using a Cole‐Parmer 500‐Watt Ultrasonic Homogenizer at 40% amplitude for 10 s on ice was used to sonicate the cell lysates, then centrifuged at 4°C for 20 min at 12,000×g. The supernatant was extracted and frozen at −80°C. A 50/50 sample to 2× SDS/PAGE sample buffer produced protein lysates. SDS/PAGE was used to separate proteins then immunoblotted using Histone H3 (D1H2) (#4499) Rabbit mAb, Acetyl‐Histone H3 (Lys9/Lys14) (#9677) Rabbit mAb, α‐Tubulin (11H10) (#2125) Rabbit mAb, Acetyl‐α‐Tubulin (Lys40) (D20G3) (#5335) Rabbit mAb, and β‐Actin (D6A8) (#8457) primary antibodies (Cell Signaling Technology, Danvers, MA). Enhanced luminol‐based chemiluminescent (ECL) was used for detection of the western blot. Quantification of bands was performed using Image J.