Las4000 gel imager

CASMCs (2 × 10⁵ cells) were cultured in SmGM‐2 medium with 10% (v/v) FBS at 37°C with 5% CO₂. Cells were fixed (15 min) in 4% (v/v) paraformaldehyde in PBS and then permeabilized with 0.5% (v/v) Tween‐20 for 10 min. For F‐actin visualization, Alexa Fluor 488–phalloidin was diluted 1:40 in 1% (w/v) BSA in PBS and then incubated with the cells for 1 h at room temperature. Cells were rinsed with PBS, counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 5 min to detect nuclei, and then examined with an InCell 6000 Imaging System (GE Healthcare, Mississauga, ON, Canada). For visualization, the Imaging System was programmed to complete whole‐well scanning of ten random visual fields from four independent plates of cells. After blocking with 5% non‐fat milk protein, membranes were incubated with primary antibody (1:1000 dilution) for 16 h at 4°C, washed extensively and then incubated with horseradish peroxidase (HRP)‐conjugated anti‐IgG secondary antibody (1:2500 dilution) for 1 h at room temperature. A final washing step was completed and then membranes were developed with enhanced chemiluminescence (ECL) reagent and imaged using an LAS4000 Gel Imager with ImageQuant densitometry software (GE Healthcare).

We performed this using the ProteoSilver
Silver Stain Kit (Sigma-Aldrich) according to its established protocol.
Briefly, we placed gels in 100 mL of fixing buffer (50 mL of ethanol,
40 mL of Milli-Q water, and 10 mL of glacial acetic acid) overnight.
Subsequently, we washed the gels for 10 min in 30% ethanol solution
and then in 200 mL of Milli-Q water for 10 min and sensitized the
gels in 100 mL of sensitization solution (1 mL of ProteoSilver sensitizer
in 99 mL of Milli-Q water). Following this, we washed the gels once
more in Milli-Q water for 10 min, equilibrated the gels in silver
solution (1 mL of ProteoSilver silver solution and 99 mL of Milli-Q
water) for 10 min, followed by two brief washes in Milli-Q water.
The gels were then developed for approximately 5 min in 100 mL of
developer solution (5 mL of ProteoSilver Developer 1, 0.1 mL of ProteoSilver
Developer 2, and 95 mL of Milli-Q water) after which the reaction
was stopped by adding 5 mL of the provided stopping solution for at
least 10 min. Stained gels were imaged using the GE LAS 4000 gel imager.