Nbp2 37931

The total protein was measured with the BCA Protein Assay Kit (Thermo Scientific). Briefly, 1.5 mg/ml of protein was used for the WES (automated western blotting, Protein Simple WES) sample preparation with 12–230 kDa cartridge kit, and the proteins were separated in WES with a capillary cartridge according to the manufacturer's protocols (Protein Simple WES, Germany). Mouse primary antibodies were used against α-SMA (Novus, NBP2-33006, 1 : 100), calponin (Sigma-Aldrich, C2687, 1 : 400), MyH11 (Santa Cruz, sc-59826, 1 : 10), and smoothelin (Novus, NBP2-37931, 1 : 100). Mouse anti-GAPDH (Novus, NB300-221, 1 : 100) served as internal control. Protein expression was normalized and quantified in relation to GAPDH and analyzed using the Compass software (Protein Simple). Protein expression on day 2 and day 4 was compared to that on day 7.

For 2D SMCs, cells were cultured on Lab-Tek chamber slides (Thermo Scientific). The indirect immunostainings for both spheroids and 2D SMCs were performed on day 2 of culture. Samples were stained at 4°C overnight using the following primary antibodies for the contractile SMC proteins: anti-α-smooth muscle actin (α-SMA) (Sigma, A5228, 1 : 200), anti-calponin (Sigma, C2687, 1 : 100), anti-smoothelin (Novus, NBP2-37931, 1 : 100), and anti-myosin heavy chain 11 (MyH11) (Santa Cruz, SC-6956, 1 : 50). The slides were incubated with a Cy3-conjugated secondary antibody (Sigma, 1 : 500) at room temperature for 1 h.
For ECM proteins, anti-collagen I (Abcam, ab260043, 1 : 150), anti-collagen III (Abcam, ab7778, 1 : 150), anti-fibronectin (Santa Cruz, sc-59826, 1 : 300), and anti-elastin (Abcam, ab9519, 1 : 100) were used. Samples were stained at 37°C for 4 h. The slides were incubated with a FITC-conjugated secondary antibody (Sigma, 1 : 500) at 4°C overnight. The slides were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Sigma, 1 : 400) and analyzed with an inverted confocal microscope (Leica DMI6000 AFC, Model SP8). For negative controls, the primary antibody was omitted. The specificity of the commercial antibodies had been thoroughly validated in our previous studies [28 (link), 29 (link)].