Protein extraction and Western blot analysis

Aortic arch and myocardial tissues (~0.1 g) were crushed in a Mikro‐Dismembrator U (Braun, Melsungen, Germany) at 2000 rpm for 60 seconds and lysed in precooled RIPA buffer (1 ml) {(100 mM Tris–HCl (pH 8), 150 mM NaCl, 5 mM EDTA (pH 8), 1 mM NaF, 1 mM Na₃VO₄, 1% Na deoxycholate, 1% Nonidet P‐40, 0.1% SDS and 1% Triton‐X100)} containing protease inhibitor cocktail (Pierce; Thermo Scientific, Winsford, UK). Total protein was quantified using the Pierce bicinchoninic acid (BCA) assay. Western blots were performed with soluble protein (10 or 20 μg), and with Hela cell lysate as a positive control. Samples from both the controls and cholesterol‐fed rabbits were loaded onto the same gels so that a direct comparison could be made. The blots were probed with the following antibodies: anti‐Hsp27 (#AF1580; R & D systems, Oxfordshire, UK), anti‐pHsp27 {(Ser82); #9709)}, anti‐p38MAPK (#9212), anti‐p‐p38MAPK (#9215), anti‐Hsp60 (#12165), GAPDH (#MCA4739; AbD Serotech, Oxfordshire, UK) and β actin (#ab8224; Abcam, Cambridgeshire, UK); all were purchased from Cell Signalling Technology Inc., (Danvers, MA, USA), except where indicated. Peroxidase‐ or phosphatase‐conjugated IgG secondary antibodies were used, and the blots were developed with enhanced chemiluminescent substrates (ECL or CPD‐star; Invitrogen, Paisley, UK).

Following analysis, the PVD membranes were stripped and reprobed with a control antibody (GAPDH or β‐actin) to allow standardization for loading. Quantification of protein band density was performed using the Syngene Gene‐Genius Bioimaging system (Cambridge, UK) and standardized to the GAPDH bands using Gene Snap and Gene Tools image analysis software.