Ma3 926

The A549 cells were lysed using RIPA buffer (Invitrogen, USA). The BCA assay (Sigma-Aldrich) was performed at a 562 nm absorbance to determine protein concentration. Protein (40 μg) was analyzed by 7.5% SDS-PAGE and then electrophoresed onto a PVDF membrane. The PVDF membrane was incubated with primary antibodies for 1 d. Primary antibodies to β-actin (1:1000, 4970S, Cell Signaling Technology, Danvers, MA, USA), TRPV6 (1:1000, ACC-036, Alomone Labs, Jerusalem, Israel), NCX1 (1:500, MA3-926, Thermo Fisher Scientific, MA, USA), and PMCA1 (1:1000, Swant, Marly, Switzerland) were used. After incubation with primary antibodies, membranes were treated with secondary antibodies (anti-rabbit, 1:3000, Cell Signaling Technology; anti-mouse, 1:3000, Cell Signaling Technology) for 1 h at room temperature. The membrane was then washed with 1× TBS-T three times each for 10 min at room temperature. An ECL solution using Chemi Doc, GenGnome 5 (Syngene, Cambridge, UK) was used to confirm antibody binding. Image J v1.37 software (Wayne Rasband, NIH, Bethesda, MD, USA) was used for target band analysis.

Atrial tissue was collected and homogenized as described previously (Alvarado et al., 2017 (link)), in a buffer containing 0.9% NaCl, 10 mM Tris-HCl pH 6.8, 20 mM NaF and protease inhibitors. Equal amounts of protein, as determined by Bradford assay, were loaded. 50 µg of tissue homogenate, in Laemmli buffer, was separated by SDS-PAGE in 4–20% TGX or AnyKD precast gels (Bio-Rad). Proteins were transferred to PVDF membrane using the iblot2 transfer system (ThermoFisher) or wet transfer. Primary antibodies were as follows: anti-RyR2 (1:2000; MA3-925, ThermoFisher), SERCA2 (1:1000; MA3-919, ThermoFisher), NCX (1:1000; MA3-926, ThermoFisher), PLN (1:5000; A010-14, Badrilla), pT17-PLN (1:5000; A010-13, Badrilla), pS16-PLN (1:5000; A010-12, Badrilla), Cav1.2 (1:200; ACC-003, Alomone), GAPDH (1:10000; MAB374, Millipore). Secondary antibodies were: goat anti-mouse-HRP (1:5000; 31437, ThermoFisher) or goat anti-rabbit-HRP (1:5000; 31463, ThermoFisher). Secondary antibody concentrations were 5x higher when using the ibind Flex system. SuperSignal ECL reagent (ThermoFisher) was used to develop membranes followed by imaging with a ChemiDoc MP apparatus (Bio-Rad). Band intensities were quantified with the ImageLab software (Bio-Rad) or using ImageJ (NIH).