Anti sm22

Cells seeded on microscope coverslips were fixed with 4% formaldehyde for 20–30 min, permeabilized with 0.4% Triton X-100 in PBS for 10–20 min, and blocked with 10% donkey serum in PBS for 1 h at room temperature. Cells were then incubated with primary antibody (diluted with 1% donkey serum in PBS) overnight at 4 °C and fluorescence-labeled secondary antibody (Invitrogen; 1:500 diluted with 1% donkey serum in PBS) at room temperature for 1 h the next day. Hoechst 33342 (Invitrogen; 1:1,000) was used to stain nuclear DNA.
Primary antibodies for immunofluorescence include anti-NANOG (Abcam, ab21624; 1:100), anti-SOX2 (Santa Cruz, sc-17320; 1:200), anti-OCT4 (Santa Cruz, sc-365509; 1:200), anti-TUJ1 (Sigma, T2200; 1:500), anti-α-SMA (Sigma, A5228; 1:200), anti-FOXA2 (CST, 8186S; 1:200), anti-Vimentin (Abcam, ab8978; 1:250), anti-α-Tubulin (Sigma, T5168; 1:500), anti-Vinculin (Sigma, V9131; 1:100), anti-ZOI (Abcam, ab96587; 1:200), anti-ClaudinV (Abcam, ab15106; 1:200), anti-SM22 (Abcam, ab14106; 1:200), anti-Calponin (Dako, M3556; 1:200), anti-vWF (Dako, A0082; 1:200), anti-eNOS (BD, 610296; 1:100), anti-VE-cadherin (CD144) (CST, 2158S; 1:100), anti-human CD31-FITC (BD biosciences, 557508, 1:100), anti-Ki67 (Vector Laboratories, ZA0731; 1:500), Phalloidin (F-actin) (Invitrogen, A22287; 1:50), anti-NF-κB P65 (RelA) (CST, 8242S; 1:200).

Smooth muscle cells were cultured in a 24-well plate at 10⁵ cells/well in 5% FCS before exposure to treatment conditions for 48 h. Cells were then scraped into equal volumes of sample buffer (Tris/HCl pH 6.8, glycerol, SDS, 2-β mercaptoethanol, bromophenol blue) and stored at − 80 °C. Samples were electrophoresed using a 12% SDS-PAGE gel, then transferred onto a PVDF membrane. This was followed by incubation in 5% milk diluted in Tris-buffered saline containing 0.1% Tween-20 (TBS-T; Bioshop, ON, Canada), then in antibodies diluted in TBS-T (anti-SMA (1:1000; mouse; Novus, ON, Canada) and anti-SM22 (1:5000; rabbit; Abcam, ON, Canada) at 4 °C overnight. This was followed by a 2 h incubation in appropriate secondary antibodies (anti-mouse HRP; 1:20,000; Fisher, anti-rabbit HRP; 1:4000; NEB, ON, Canada). Membranes were then exposed to a chemiluminescent substrate (Luminata Forte) and imaged using a ChemiDoc MP System (Bio-Rad, ON, Canada). Images of bands exposed until just below band saturation were quantified using Image Lab software (Bio-Rad). The membrane was then reprobed with anti-GAPDH antibodies (1:5000) as above for normalization to loading volume.