Acetyl histone h3 lys9 lys14 antibody

Fibers were PFA-fixed and Triton-permeabilized as described above (Nuclear organization of single fibers). Fibers were treated with mouse-on-mouse block (Vector Laboratories, BMK-2202) for 1 hour, blocked in 10% goat serum in PBS (Sigma-Aldrich, G9023) for 30 minutes, and treated with primary antibodies diluted in goat serum blocking buffer. Prelamin A was visualized using as primary antibody Santa Cruz Biotechnology Inc. sc-6214, c-20 (secondary antibody: Alexa Fluor 488, Invitrogen, A-11078) and Pax7 using Developmental Studies Hybridoma Bank (DSHB) AB 528428 as primary antibody (secondary antibody: Alexa Fluor 488, Invitrogen, A-11001). Lamin A (Abcam, ab8980) and nesprin-1 (Abcam, ab192234) were matched with Alexa Fluor 488 and 594, respectively (Invitrogen, A-11001 and A-11012). Acetyl-histone H3 (Lys9/Lys14) antibody (Cell Signaling Technology, 9677) matched with Alexa Fluor 594, respectively (Invitrogen, A-11012).

SIRT1 catalytic activity was assessed by quantifying the acetylation of one of its substrates, H3K9/14, as previously reported (Bugyei‐Twum et al. 2018). In brief, total protein was extracted with ice‐cold radioimmunoprecipitation buffer containing a protease inhibitor mixture and quantified with a Bio‐Rad Protein Assay Reagent. Protein samples were then separated by SDS‐PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk in TBS‐T and probed with the acetyl‐histone H3 (Lys9/Lys14) antibody (Cell Signaling Technologies #9677, Danvers, MA). Anti‐tubulin antibody conjugated to horseradish peroxidase (Cell Signaling Technologies) was used as a loading control so that abundance of acetyl‐histone H3 (Lys9/Lys14) was quantified by enhanced chemiluminescence detection system (Amersham Biosciences, Amersham, UK) relative to tubulin.