Peroxidase conjugated anti rabbit or anti mouse antibodies

Following exposure to PQ, the cells were detached from the culture dishes with a cell scraper and collected together with floating cells by centrifugation. The collected cells were disrupted using a sonication device (10 s, 3 times; Sonifer 150, Branson, Danbury, CT, USA) in STE buffer [0.32 M sucrose, 10 mM Tris–HCl (pH 7.4), 5 mM EDTA, 50 mM NaF, 2 mM Na₃VO₄] containing protease inhibitor cocktail (Complete, Roche, Manheim) on ice. Protein concentrations were determined by the Coomassie Brilliant Blue staining method [18 (link)]. Equal amounts of proteins were separated by SDS-PAGE according to the method of Laemmli [19 (link)] and transferred to a PVDF membrane. For the analysis of conditioned medium, equal volumes of medium were loaded and separated by SDS-PAGE. After blocking, the membrane was incubated overnight at 4°C with appropriate primary antibodies, washed with TBS-Tween and incubated for 45 min with peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Promega, Madison, WI, USA). Bands were detected using a Western Lightning Chemiluminescence Reagent Plus Kit (PerkinElmer Life Science, Boston, MA, USA), and the intensities of the bands were quantified using an image analyzer (CS analyzer; Atto, Tokyo, Japan).

Right atrial (RA) and left atrial (LA) tissue homogenates were probed with the following antibodies: gp91^phox (BD Transduction); total and phosphorylated RyR2 at serine-2808 and serine-2814 (ThermoFisher Scientific); α-smooth muscle actin (α-SMA; Cell Signalling); vimentin (Exbio); HRP-conjugated GAPDH (Sigma Aldrich); and total CaMKII (Cell Signalling) and an antiserum to oxidized CaMKII that was kindly provided by Dr ME Anderson, Johns Hopkins University, USA. Detection of primary antibodies was performed using peroxidase-conjugated anti-rabbit or anti-mouse antibodies (both Promega, USA). GAPDH was used as a loading control except for phosphorylated protein levels which were normalized by the respective total protein levels. All western blots were run under reducing conditions apart from those to detect oxidized CaMKII, which was performed under non-reducing conditions to preserve the redox modification. For the detection of RA and LA NOX2, atrial tissues were pooled from two different animals prior to homogenization.