Alphaease fc imaging system

Neuronal cells were grown and treated in 6-well plates. Total proteins were extracted with a Nuclear Extraction Kit (Active Motif, Carlsbad, CA, USA) and their concentration determined by using the BCA protein assay kit (Pierce Biotechnology Inc., Rockford, AZ, USA). Fifteen µg of protein were loaded onto a 12% SDS-polyacrylamide gel. After electrophoretic separation (125 V, for 1 h 30), proteins were transferred onto PVDF membranes (0.22 μm pore size, BioRad) at 25 V overnight. The membranes were blocked in TBST (TBS + Tween 0.05%) with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies: anti-cleaved caspase-3 antibody (1:500) anti-cleaved PARP-1 antibody (1:500) or an anti-β-actin antibody (1:500) (Cell Signaling, Danvers, MA, USA). The blots were then rinsed three times with TBS 0.1% Tween 20 and incubated with peroxidase-conjugated (POD) secondary antibody (1:10,000) for 2 h at room temperature. Blots were washed with TBS 0.1% Tween 20 three times and the signal finally revealed by enhanced chemiluminescence with the AlphaEase FC imaging system (Alpha Innotech, San Leandro, CA, USA) and analyzed with AlphaEase FC software (Alpha Innotech) and ImageJ (imagej.nih.gov).

Neuronal cells were seeded at 30,000 cells/cm², differentiated and treated in collagen-coated 6-well plates. Total proteins were extracted (Nuclear Extraction Kit, Active Motif, Carlsbad, CA, USA) and concentrations were determined by bicinchoninic acid quantification (BCA protein assay kit, Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of protein were loaded onto a 12% SDS-polyacrylamide gel. After electrophoretic separation (125 V, for 1.5 h), proteins were transferred onto PVDF membranes (0.22 μm pore size, BioRad) at 25 V overnight. The membranes were blocked for 30 min to 1 h and incubated overnight at 4 °C with primary antibodies anti-Bax, anti-Bcl-2, anti-LC3, anti-p62 (Progen Biotechnik GmbH, GP62-C) and anti-actin, (1:200, 1:100, 1:500, 1:1000, and 1:2000, respectively). The blots were then incubated with the appropriate peroxidase-conjugated secondary antibody (1:10,000) for 2 h at room temperature and finally developed with an enhanced chemiluminescence substrate solution (ThermoFisher Scientifics, Ottawa, ON, Canada). Immunopositive chemiluminescent signals were visualized with the AlphaEase FC imaging system (Alpha Innotech, San Leandro, CA, USA) and analyzed using AlphaEase FC (Alpha Innotech San Leandro, CA, USA) and ImageJ (https://imagej.nih.gov/ij/) software packages.

Western blot was performed as described in our previous publication (57 ). Proteins were extracted from cells using the lysis buffer containing protease/phosphatase inhibitor (Sigma–Aldrich). Equal amount of proteins (30 μg per lane) were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore), and probed with primary antibodies at 4 °C overnight (anti-Orai1, 1:800; anti-STIM1, 1:2000; anti-nephrin, 1:1000; anti-synaptopodin, 1:800; anti-podocin, 1:1000; anti-GAPDH, 1:300; and anti-α-tubulin, 1:200). After washing membranes with Tris-buffered saline with Tween for three times, the membranes were then incubated with secondary antibodies (1:5000) in room temperature (RT) for 1 h. Orai1 protein band was developed with Super Signal West Femto, but all other protein bands were developed with Pico Luminol/Enhancer Solution (Thermo Scientific). The bands were visualized and captured using the AlphaEase FC Imaging system (Alpha Innotech). The band density was measured by AlphaEase FC software (Alpha Innotech) as previously described (57 ).