Restoretm plus western blot stripping buffer

Proteins were extracted in RIPA buffer supplemented with a cocktail of proteases and phosphatases inhibitors (all from Sigma) and concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Primary antibodies (online supplementary Table S3) were incubated for 16 h at 4°C. Either β‐actin or β‐tubulin were used as loading control. Anti‐rabbit or ‐mouse IgG HRP‐conjugated were employed as secondary antibodies (both 1:5000, GE Healthcare). Membrane development was performed by an enhanced chemiluminescence‐based detection method (ECL™ Prime Western Blotting Detection Reagent, GE Healthcare) in a ChemiDoc‐MP system (Bio‐Rad). No more than one stripping procedure was performed on an individual membrane (RestoreTM Plus Western Blot Stripping Buffer, Thermo Fisher Scientific).

Primary human arterial endothelial cells and PASMC were lysed in RIPA buffer (Sigma-Aldrich) and 10 μg of protein were run on a 10% SDS polyacrylamide gel, followed by electrotransfer to an Amersham Hybond P 0.45 PVDF Blotting Membrane (GE Healthcare, Wien, Austria). After blocking with 5% non-fat dry milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20), the membrane was incubated overnight at 4°C with one of the following antibodies: anti-p38, anti–phospho-p38 (T180/Y182), anti-c-Jun; anti-phospho c-Jun (S73), anti-phospho Erk1/2 (T202/Y204), anti-Erk1/2, anti-phospho c-Fos (S32) all from Cell Signaling and anti-c-Fos (Novus Biologicals, Cambridge, UK) were used at dilution of 1:1000; and anti-TLR4 (Abcam), anti-RAGE (1:500; Abcam) and rabbit anti–alpha-tubulin (1:5000; Cell Signaling, Boston, MA, USA). After washing, the membranes were incubated with horseradish-peroxidase–labelled secondary antibodies (1:5000, anti-rabbit-HRP, Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and proteins detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare). Antibodies were removed by incubating the membrane for 15 min. with stripping buffer (RestoreTM PLUS Western Blot Stripping Buffer; Thermo Scientific). Densitometric analysis was performed with ImageJ (National Institutes of Health, USA).