Ecl western blotting detection reagent kit

On the day of the analysis, cell lysates were thawed on ice and then combined with 2x Laemmli loading Buffer (BioRad) with 0.05% BME. The samples were heat denatured for 10 min at 95 °C and loaded into a Bolt™ 4 to 12% (vol/vol), Bis-Tris precast polyacrylamide gels (Invitrogen by Thermo Fisher). Gels were run at 120V in 1x MOPS Buffer and were then transferred to a 0.2 μm PVDF nitrocellulose membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad) using the Trans-Blot Turbo Transfer System (13A, 25V, 7 min) (BioRad). The membrane was blocked in 5% BSA for 1 h followed by overnight incubation at 4 °C with primary antibodies (1:1000 anti-mCherry [Cell Signaling Technology] or 1:1000 anti-DYKDDDDK [Cell Signaling Technology]). Membranes were washed with TBST (3x, 5 min) and incubated with secondary anti-rabbit HRP antibody for 1 h at room temperature followed by a subsequent washing with TBST (3x, 5 min). The membrane was developed with ECL western blotting detection reagent kit (Thermo Scientific) and imaged on ChemiDoc XRS+ with Image Lab Software (Bio-Rad).

Cell pellets were lysed in DPBS and complete protease inhibitor, EDTA-free using a probe sonicator (Branson Sonifier model 250) with 15 pulses (30% duty cycle, output setting = 4). Proteins were resolved by SDS–PAGE, transferred to 0.45 μM nitrocellulose membranes (Thermo Fisher) which were blocked with 5% milk in TBST buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl with 0.1% tween 20]. Primary antibodies were used at the following concentrations: 1:2,000 anti-Na⁺/K⁺-ATPase (Cell Signaling, no. 3010), 1:2,000 anti-FLAG-HRP conjugated (Abcam, ab2493), 1:2,000 anti-TMEM164 polyclonal (Thermo Scientific, PA5-58540). Blots were incubated with primary antibodies in 5% milk in TBST at 4°C overnight. Following another TBST wash (5 times, 5 min), membranes were incubated with 1:5,000 anti-rabbit IgG, HRP conjugated secondary antibody in 5% milk in TBST (Cell Signaling, no. 7074S) at room temperature for 1 h. Membranes were washed with TBST (5 times, 5 min) and developed with ECL western blotting detection reagent kit (Thermo Scientific) and detected with Bio-Rad ChemiDoc MP imaging system.