Anti vinculin

Proteins were isolated with a lysis buffer composed of 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 0.1% SDS and the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltman, MA, USA). The BCA protein assay kit (Thermo Fisher Scientific) was used to quantify the protein content. An equal amount of protein from each sample was separated on CriterionTM Precast Gel Tris-HCl (Biorad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA), as previously described^{55 (link)}. The membranes were blocked for 2 hours in 5% non-fat dry milk PBS with 0.1% Tween 20 (Sigma-Aldrich) at room temperature and incubated overnight at 4 °C with primary antibody. After washing, the membranes were incubated for 1 hour at room temperature with horseradish-peroxidase-conjugated secondary antibody. The following primary antibodies were performed: anti- Collagen IV (1:1000, Abcam), anti-Fibronectin (1:1000, Abcam), anti-Vitronectin (1:1000, Abcam), anti-CASP3 (1:1 000, Cell Signaling Technology), anti-CASP9 (1:500, Cell Signaling Technology), anti-BAX (1:1000 Cell Signaling Technology), anti-RHO (1:1000 Merck Millipore), anti-Vinculin (1:1000 Thermo Fisher Scientific). Vinculin was used as the loading control.

A375, A375-GFP-PMCA4b, A375-GFP-PMCA4b-LA, MCF-7, MCF-7-GFP-4b, and MCF-7-Sh-PMCA4b cells were cultured in a 6-well plate for 48 h. The total protein content of the cells was precipitated with 6% TCA. Samples were separated by using 10% or 15% acrylamide gels, as appropriate, and electroblotted onto PVDF membranes (Biorad, Hercules, CA, USA), as described previously [28 (link)].
Blots were immunostained with the following rabbit monoclonal primary antibodies: antivinculin (1:100, ThermoFisher scientific, cat. # 700062), anti-P-cofilin (Ser3) (1:1000, Cell Signaling Technology, Danvers, MA, USA, cat. # 77G2), rabbit polyclonal antibody: anti-β-tubulin (1:1000, Abcam, cat. # ab6046), anti-PMCA1 (1:1000, Affinity BioReagents, cat. # PA1-914), mouse monoclonal antibodies: anti-PMCA4 (JA9, 1:1000, Sigma-Aldrich, cat. # P1494), anti-NA⁺/K⁺ ATPase (1:2000, Enzo Life Sciences, cat. # BML-SA247), and chicken polyclonal antibody: anti-GFP (1:5000, Aves, GFP-1020). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-chicken secondary antibodies were used for detection (Jackson ImmunoResearch, dilution 1: 10,000) and were visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific). The ImageJ software was used for densitometry analysis.