Mouse monoclonal anti α smooth muscle actin antibody

Cells were lysed in 20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, and 100 mg/mL-1 phenylmethylsulphonyl fluoride. Total cell extracts were cleared by centrifugation (10000 g for 20 min at 4°C). 40 mg of protein were analysed in 10% sodium dodecyl sulphate–polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane. Western blot analysis was performed using anti- smooth muscle β-actin mouse monoclonal antibody (1:5000, Cell Signalling), anti-smooth muscle α-actin mouse monoclonal antibody (1:1000, Sigma), anti- myosin (smooth) clone HSM-V mouse monoclonal antibody (1:1000, Sigma) and anti-desmin rabbit polyclonal antibody (1:1000, Sigma). Membranes were then incubated with horseradish peroxidase conjugated anti-rabbit IgG (1:3000, Cell Signalling) or anti-mouse IgG (1:3000, Cell Signalling), and signals visualised by enhanced chemoluminescence (ECL).

Cells were plated onto glass slides, which were resided in culture plates and treated as indicated above. After the treatment, cells were fixed with PBS-3% formaldehyde and permeabilized with PBS-1% Triton X-100, blocked in PBS-0.1% Tween 20- 3% BSA and incubated with anti-smooth muscle α-actin mouse monoclonal antibody (1:1000, Sigma), anti-myosin (smooth) clone HSM-V mouse monoclonal antibody (1:1000, Sigma) and anti-desmin rabbit polyclonal antibody (1:1000, Sigma), followed by incubation with an anti-mouse IgG or anti-rabbit IgG antibodies conjugated with CY3. Finally, coverslips were mounted on Vectrashield solution containing DAPI for DNA staining and the percentage of the cells that expressed α-actin, SM-MHC (Smooth muscle-Myosin Heavy Chain) and desmin proteins was estimated.

Bladder SMC were fixed with 3% paraformaldehyde solution at 22–24°C (RT) for 10 min, treated with 0.1 M glycine in phosphate‐buffered saline (PBS) for 5 min, and incubated in blocking solution (PBS containing 0.1% Triton X‐100 and 1% bovine serum albumin) for 1 h at RT. Subsequently, cells were incubated overnight at 4°C with goat polyclonal anti‐HERG antibody (dilution 1:100), which had been validated previously using overexpression systems (Barrese et al., 2017 (link)) now discontinued or with mouse monoclonal anti‐HERG antibody (1:100; sc‐377388), and mouse monoclonal anti‐α‐smooth muscle actin antibody (dilution 1:1000) (Cat.#: A5228; Sigma). Samples were washed with PBS and incubated for 1 h with donkey antigoat secondary antibody conjugated to Alexa Fluor 568 (Cat.#: A‐11057) and donkey antimouse secondary antibody conjugated to Alexa Fluor 488 (Cat.#: A32766) (dilution 1:100) (ThermoFisher). All antibodies were diluted in the blocking solution. No primary antibody was used as a negative control. Coverslips were mounted on glass slides; images were acquired using a Nikon A1R confocal microscope (Nikon Instruments Europe BV).