Integrin α2

Aortic SMC were fixed at 24 hrs after plating by immersion in 2% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS). Cells were then washed in a glycine buffer and incubated overnight at 4 °C with primary antibodies against integrin α2 (Abcam, San Francisco, CA, USA), integrin α5 (Milipore Sigma, Burlington, MA, USA), and smooth muscle α-actin (SMα-actin) (Millipore Sigma, Burlington, MA, USA) diluted in a sodium citrate buffer containing BSA and Triton X [24 (link)]. After washing, cells were incubated with Alexa 568 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature, washed again and immediately imaged in DPBS. A similar procedure with overnight incubation was followed for the primary antibody against integrin β1 or β3 both pre-conjugated with Alexa 488 (BioLegends, San Diego, CA, USA). For smooth muscle γ-actin (SMγ-actin, Actg2) staining, cells were first fixed with 1% paraformaldehyde in DPBS followed by permeabilization with cold methanol [25 (link)]. Staining was performed as described by using an SMγ-actin primary antibody [26 (link),27 (link)] followed by Alexa 488 secondary antibody (Jackson Immuno Research, West Grove, PA, USA).

For immunofluorescence, antibodies and the probes used were as follows: F-actin (phalloidin–Alexa Fluor 488, catalog A12379 or phalloidin–Alexa Fluor 647, catalog A22287, Invitrogen); nuclei (NucBlue Fixed Cell Stain Ready Probes, catalog R37606, Invitrogen); aggresome (ProteoStat, catalog ENZ-51038, Enzo Life Sciences Inc.); UNC45A (catalog sc-101493, 1:50, Santa Cruz Biotechnology Inc.); villin (catalog sc-58897, 1:50, Santa Cruz Biotechnology Inc.); DRA (catalog ab83545, 1:100, Abcam); Rab 11 (catalog 71-5300, 1:25, Invitrogen); NHE3 (catalog NBP-82574, 1:500, Novus Biologicals); MYO5B (catalog HPA069773, 1:400, Sigma-Aldrich); Epcam (catalog AF960, 1:100, R&D); CFTR (catalog ab59394, 1:100, abcam); NA/K-ATPase (catalog MA5-32184, 1:100, Thermo Fisher); integrin-α2 (catalog ab181548, 1:100, Abcam); and E-cadherin (catalog 13-700445, 1:100, Thermo Fisher). For STED, F-actin (phalloidin, phalloidin-ATTO 550, Invitrogen) was used. For Western blot, the following antibodies were used: actin (catalog 5125, 1:1000, Cell Signaling); FLAG (catalog F3165, 1:1000, Sigma-Aldrich); GAPDH (catalog 14C10, 1:1000, Cell Signaling Technology); HSP90 (catalog 4874, 1:1000, Cell Signaling Technology); UNC45A (catalog 171328, 1:500, Abcam); and MYO5B (catalog PA5-49519, 1:500, Thermo Fisher).