Acetyl α tubulin lys40 d20g3

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.

Total protein lysates were generated using RIPA buffer (Sigma Aldrich, St. Louis, MO, USA) according to manufacturer’s instructions, supplemented with protease inhibitors, and separated on 10% TGX gel (Bio-Rad, Hercules, CA, USA) under reducing conditions. The gel was transferred to a PVDF membrane and probed with 1:3000 class III β-tubulin (TUJ1; BioLegend, San Diego, CA, USA), 1:10,000 GAPDH (Cell Signaling Technologies, Danvers, MA, USA), Cofilin (D3F9) (1:1000, Cell Signaling Technologies), p-Cofilin (Ser3) (1:1000, Cell Signaling Technologies), monoclonal α-tubulin (DM1A) (1:2000, Sigma Aldrich), anti-detyrosinated α-tubulin (glu-tubulin) (1:1000, Abcam, Waltham, MA, USA), tyrosinated α-tubulin (1:2000, Sigma Aldrich), acetyl α-tubulin (Lys40, D20G3) (1:1000, Cell Signaling Technologies), α-Actin (AC-15) (1:10,000, Sigma Aldrich, Burlington, MA, USA), CAPZB (1:1000, Bio-Rad), and Diap1 (1:1000, Cell Signaling Technologies). Standard molecular weight markers were used (Precious Plus Protein Standards #1610374/ #1610375, BioRad, St. Louis, MO, USA). Densitometry was analyzed with ImageJ: https://imagej.nih.gov/ij/ (accessed on 11 June 2018). Experiments were performed in duplicate and representative images are shown. Raw western blot figures can be found in the supplementary materials.