Rabbit anti alpha smooth muscle actin

Cells of 3–6th passage were attached to L-polylysine-coated glass slides, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Bio-Rad, Hercules, CA, USA) for 20 min at room temperature, and then blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h, and incubated with mouse anti-CD90 (1:100, cat. no. ab134358; Abcam, Cambridge, UK), rabbit anti-alpha smooth muscle actin (1:100, cat. no. A17910; ABclonal, Wuhan, China), mouse anti-fibronectin (1:100, cat. no. ab6328; Abcam, Cambridge, UK) monoclonal antibody conjugated with fluorochrome for 12 h at 4 °C. Then, the cells were incubated for 40 min at room temperature with secondary antibodies (goat anti-mouse IgG conjugated with Alexa Fluor 488, 1:400, cat. no. A11029; Thermo Fisher Scientific, Waltham, MA, USA and goat anti-rabbit IgG conjugated with Alexa Fluor 488, 1:400, cat. no. ab150077; Abcam, Boston, MA, USA) and washed. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, UK). The actin–cytoskeleton of the cells was stained with phalloidin coupled with Alexa Flour 488 (1:200, cat. no. A12379, Thermo Fisher Scientific, Waltham, MA, USA) for 60 minutes, as per the manufacturer’s instructions. The cells were photographed using an Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).

Western blot analysis was carried out to confirm the proteomics data and to investigate the effect of the cytokine/hormone treatments on the level of the protein of interest. For our Western blot analysis the protocol of Towbin et al. [26 (link),27 (link)] was adapted. First, 50 µg of the cell extracts or secretome proteins were subjected to a denaturation step with Laemmli buffer and loaded on a 12% SDS-gel. Rabbit anti-fibronectin (FN1) (Sigma), rabbit anti-alpha smooth muscle actin (Abcam), mouse anti-peptidyl-prolyl cis-trans isomerase A (PPIA) (Abcam), and mouse anti-β-actin (Sigma), as the primary antibodies were diluted in blocking buffer, then added to the membrane and allowed to incubate overnight at 4 °C. Molecular Probes^® Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 µm resolution on a Fuji FLA-5100 scanner (Fuji Photo, Kanagawa, Japan) with single laser emitting excitation light.