Ab77804

Protein extracts were prepared by lysing the cells in RIPA buffer containing protease inhibitors (cOmplete, Mini, EDTA free; Roche). Protein yield was quantified using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE before wet transfer onto PVDF membrane (GE Healthcare). Non-specific binding sites were blocked by 5% non-fat dry milk in Tris-buffered saline Tween [130 mM NaCl, 20 mM Tris (pH 7.6) and 1% Tween 20] for 1 h at room temperature on a rolling/shaking surface. The following primary antibodies were purchased from Abcam: anti-CNN1 (ab46794, 1:1000), anti-ELN (ab77804, 1:500) and anti-β-actin (ab8226, 1: 100,000). Anti-VAP-1 antibodies were purchased from R&D systems (MAB3957, 1:1000) and the anti-MYH11 antibodies were purchased from Proteintech (21404-1-AP, 1:1000). Protein complexes were visualized with ECL prime chemiluminescence (GE Healthcare) and imaged using the G:BOX from Syngene.