Figure 3: Elevation of MRTF-A Acetylation and Total Protein by HDAC6 Inhibition in Cultured Cells and Rat Artery Explants

(A) Western blotting of myocardin-related transcription factor A (MRTF-A) total protein. Starved (overnight) MOVAS cells were pretreated with vehicle or HDAC inhibitors for 2 h and then stimulated with PDGF-BB for 48 h, as described in Figure 1A. MRTF-A duplicate bands are generally observed in the literature (25). Data are quantified as fold changes versus control (vehicle + PDGF, normalized value as 1); mean ± SEM; n = 3 independent experiments; *p < 0.05 compared with control (1-sample Student’s t-test). (B) Western blotting of acetylated MRTF-A. Starved MOVAS cells were incubated with vehicle or 5 μmol/l tubastatin A for 24 h and then collected for immunoprecipitation (IP) by using an MRTF-A antibody or equal amount of immunoglobulin G (IgG) for control. Immunoblotting (IB) was performed to detect acetyl-lysine. Dashed line separates IP from Input, both loaded on the same gel. Data are quantified as fold changes versus IgG control (normalized value as 1); mean ± SEM; n = 3 independent experiments; *p < 0.05 compared with control (1-sample Student’s t-test). (C, D) Co-immunoprecipitation (Co-IP) of HDAC6 with MRTF-A. HDAC6 was overexpressed in HEK293 cells. Equal amounts of IgG and MRTF-A antibody were used for IP. Co-immunoprecipitated proteins were detected by IB for (C) HDAC6 or for (D) MRTF-A to confirm the functional specificity of the MRTF-A antibody (Ab). Presented are blots from 1 of 3 similar experiments. (E, F) Western blotting of acetylated MRTF-A and its total protein, respectively. Shown are representative blots from 1 of 3 similar experiments. For ex vivo treatment of arteries with tubastatin A, rat aortas deprived of endothelium were cut into strings and cultured in Dulbecco’s modified Eagle’s medium/F12 containing 0.5% FBS. After incubation in the presence of vehicle or 10 μmol/l tubastatin A for 24 h, the artery explants were (E) pooled and homogenized for IP and then IB or (F) directly used for IB. Dashed box indicates shorter exposure as opposed to longer exposure of the same blot (upper, indicated by arrow). Dashed blue line separates Input and IP on the same blot. Abbreviations as in Figures 1 and 2.

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