Sc 33766

Protein samples were separated by SDS–PAGE (10%), blotted onto nitrocellulose membranes and analyzed by chemiluminescence-based immunodetection according to standard procedures. The following primary antibodies were used for incubation at 4°C for 24 h: ms-anti-NFAT5 1:500 (sc-398171, Santa Cruz), rb-anti-histone H3 1:1000 (ab1791, Abcam), rb-anti-alpha-Tubulin 1:1000 (2144, Cell Signaling), ms-anti-DDK 1:1000 (TA50011-100, Origene), ms-anti-SMMHC 1:1000 (14-6400-80, eBioscience), rb-anti-calponin 1:2000 (ab46794, Abcam), or rb-anti-myocardin 1:1000 (sc-33766, Santa Cruz), ms-anti-beta-actin 1:5000 (ab6276, Abcam).

VSMCs were seeded on glass coverslips in 24-well cell culture plates at a density of 10³ cells/cm², and then incubated overnight in normal cell growth conditions. For immunofluorescence staining, cells were washed with PBS for 3 times followed by fixation and permeabilization in 75% acetone in ethanol for 5 min. Cells were then blocked with 10% goat serum for 1 h and incubated overnight with primary antibodies against SMYD2 (# 9734, Cell Signaling, RRID: AB 10889559), SM α-actin (ab5694, Abcam, RRID: AB 2223021), myocardin (sc-33766, Santa Cruz, RRID: AB 2148748), myocardin-related transcription factor-A (MRTF-A: sc-398675, Santa Cruz, RRID: AB 2142498), and MRTF-B (PA5-37105, Thermo Fisher Scientific, RRID: AB 2553910). Cells were then rinsed with PBS for 3 times and incubated with Alexa Fluor 488 or 568-conjugated secondary antibodies (1:400 dilution, Thermo Fisher). Finally, cells were counter-stained with DAPI (1:10,000 dilution, Sigma) and mounted with mounting medium. Fluorescence images of stained cells were captured via using a Leica immunofluorescence microscope.