Rabbit anti vinculin

An indirect immunofluorescence method was utilized on cells fixed in paraformaldehyde. The primary antibodies were rabbit anti-MAP2 (Merck Millipore, Billerica, MA, USA), rabbit anti-vinculin (Sigma-Aldrich Co.), and rabbit anti-β-tubulin III (Sigma-Aldrich Co.). Alexa Fluor 488 donkey anti-rabbit antibody was applied as secondary antibody (Invitrogen; Thermo Fisher Scientific), and DAPI was utilized as nuclear counterstain. F-actin was detected by phalloidin-FITC or phalloidin-rhodamine (Sigma-Aldrich Co.). Samples were observed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Nuclei were stained with DAPI (Sigma-Aldrich Co.).

The right lung was frozen, pulverized, and diluted in radioimmunoprecipitation assay buffer (1% Triton X‐100, 0.24M sodium deoxycholate, 0.35M sodium dodecyl sulfate in 1× Tris buffered saline) with protease and phosphatase inhibitors. Lung lysates were used for Western blot assays. The following primary antibodies were used: rabbit anti‐AT₁R and anti‐AT₂R (1:1,000; Novus Biologicals), rabbit anti–β‐actin (1:5,000; Sigma), rabbit antivinculin (1:2,000; Sigma), mouse anti‐eNOS (1:1,000; BD Pharmingen), mouse anti–phosphorylated eNOS Ser¹¹⁷⁶ (1:1,000; BD Pharmingen), mouse anti–estrogen receptor α (ERα) (1:1,000; R&D), rabbit anti‐ERβ (1:1,000; ThermoFisher Scientific), and mouse anti‐GAPDH (1:1,000; Biolegend).
Bound antibodies were visualized by chemiluminescence with a Luminata Forte Western HRP Substrate (Merck KGaA) using either a horseradish peroxidase–conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibody. Band intensity was analyzed using ImageJ, and results were normalized to the expression of β‐actin, GAPDH, or vinculin, as loading controls.