α tubulin 11h10

Isolated 1–3 × 10⁶ PBMCs and 0.5–1 × 10⁶ human vascular endothelial cells were pelleted and treated with homemade lysis buffer [48 (link)]. This is succeeded by measuring the protein concentration of the samples at 562 nm with a spectrometer (Tecan, Megelan, using Pierce^TM BCA Protein Assay Kit (Cat#23225, Thermo Fischer Scientific), where 20 µg of sample protein was subjected to SDS page electrophoresis and transferred to the PVDF membrane according to the standard laboratory procedure. We employed human primary antibodies for phospho-STAT-3 (Tyr 705) (D3A7), STAT-3 (D3Z2G), phospho-P65 (93H1), P65 (D14E12) (1:1000 or 1:2000), and cleaved caspase-1 (p-20) (Bally-1, Adipogen Life Sciences, San Diego, CA, USA) to detect the respective proteins. We used α-Tubulin (11H10) (Cell Signaling, Danvers, MA, USA) and/or β-actin (2D4H5) (Proteintech, Rosemont, IL, USA) as the loading protein and anti-rabbit IgG, HRP-linked antibody (Cat# 7074, Cell Signaling), and AP-conjugated anti-mouse IgG (S3721, Promega, Madison, WI, USA,) were used as secondary antibodies. As substrate, we used Pierce^TM ECL Western, Super Signal^TM West Femto, or BCIP/NBT (Promega). The subsequent development of bands was detected using an INTAS ECL ChemoStar Imager (INTAS science Imaging) and further densitometries were quantified using LabImage ID software.

FDLE cell inserts were placed on ice, and protein lysates prepared and analyzed as described elsewhere^{30 (link)}. Phosphorylation of AKT was analyzed using antibodies against phospho-AKT at Ser473 (# 9271, Cell Signaling Technology, Frankfurt am Main, Germany), and AKT (# 9272, Cell Signaling Technology, both kindly provided by A. Garten). Phosphorylation of ERK1/2 (extracellular-signal regulated kinases) was analyzed using antibodies against phospho-ERK1/2 at Tyr202/Tyr204 (# 9101, Cell Signaling Technology), and ERK1/2 (# 9102, Cell Signaling Technology, both kindly provided by A. Garten). EGFR (C74B9, # 2646, Cell Signaling Technology) expression was measured and α-Tubulin (11H10, # 2125, Cell Signaling Technology) expression was used as a reference.

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.