Serca

The primary antibodies sarco/endoplasmic reticulum calcium ATPase (SERCA, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #271669), C/-EBP homologous protein (CHOP, 1:100, Santa Cruz Biotechnology, #7351), Bcl-2 (1:500, Cell Signaling Technology, Danvers, MA, USA, # 4223S), p-PERK (1:500, Abcam, Cambridge, UK, #156919), cytochrome c (1:100, Abcam, #90529), and β-actin (1:2000, Santa Cruz Biotechnology, #47778) were purchased and maintained overnight at 4 °C. Same quantities of protein were divided on 8–10% SDS (sodium dodecyl sulfate-polyacrylamide) gels and electro-transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Process of blocking was performed with 10% non–fat milk in TBS–T for 1 h at RT (room temperature). The membranes were then washed 3 times with TBS-T and probed with the corresponding secondary antibodies conjugated to HRP (horse radish peroxidase, Santa Cruz, CA, USA) at RT for 1 h. After washing, the blots were developed with ECL reagents (Pierce) and exposed using Kodak X–OMAT AR Film (Eastman Kodak, Rochester, NY, USA) for 1–5 min.

For intracellular staining of endogenous TRPC3 or NFκB-p65, cells were fixed with 4% paraformaldehyde for 20 min in LabTek II chambers and then permeabilized with 0.1% Triton X-100 for 20 min at room temperature. Cells were treated with 5% goat serum for 30 min and incubated with antibodies against TRPC3 (Alomone Labs, validated in TRPC3-deficient cells), NFκB-p65 (Cell Signaling), or SERCA (Santa Cruz) for 1 h. After washing, the cells were incubated with spectrally appropriate Alexa Fluor-conjugated Fab fragments against primary species antibodies (Molecular Probes) for 1h. After intracellular staining, nuclei were counterstained with the DNA-binding dye Hoechst 33342 (Invitrogen). All images were captured with a Leica confocal system TCS SP5X inverted microscope with a HCS Plan Apo CS 63X/1.4 NA oil immersion lens. Leica Application Suite Advanced Fluorescence software was used for the capture, and ImageJ was used for deconvolution and image presentation. Translocation was analyzed using Cell Profiler software (Broad Institute) with a custom pipeline (available upon request) that automatically detects cell boundaries and calculates the ratio between cytoplasm and nuclei fluorescence median.