Criterion transfer system

The Bicinchoninic acid protein assay (Thermo Fisher Scientific) was performed to determine protein concentration of cellular lysates. Fifty micrograms of total protein from cellular lysates was prepared in Laemmli sample buffer and then loaded onto 4–20% Tris·HCl polyacrylamide gels. The proteins were resolved on the Criterion electrophoresis system (Bio-Rad) before being electrically transferred to nitrocellulose blotting membranes (Thermo Fisher Scientific) using the Criterion transfer system (Bio-Rad). A solution of 5% nonfat milk in 1× Tris-buffered saline (TBS; Bio-Rad) was used to block the membranes for 1 h at room temperature. The membranes were then washed with 1× TBS and then incubated with primary antibodies (ENaCα [43 (link)], ENaCβ [43 (link)], ENaCγ [44 (link)], NPRC antibody [5 (link)] at a dilution of 1:1000 in 5% BSA 1× TBS (wt/vol) while on a rocker at 4 °C overnight. Membranes were washed with 1× TBS before being incubated for 1 h in secondary antibody solution containing horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at a dilution of 1:3000 prepared in blocking solution. Membranes were washed with 1× TBS, incubated with SuperSignal Dura Chemiluminescent Substrate, and then imaged on a Bio-Rad ChemiDoc MP Imaging System with Image Lab Software Version 6.1.0 Build 7 (Bio-Rad).

HEK293FT cells transfected as for BRET assay, THP1 cells, or human primary monocyte-derived dendritic cells were lysed as described previously [44 (link)]. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher) and equalized between samples. For Venus immunoprecipitation, Venus cross-reacting GFP-Trap beads (Chromotek) were used according to manufacturer’s instructions. Samples and Novex Sharp protein size marker (Thermo Fisher) were separated by SDS-PAGE using the NuPAGE system with 4–12% Bis-Tris gels (Thermo Fisher) and transferred onto nitrocellulose membranes using the Criterion transfer system (Bio-Rad). Membranes were probed with antibodies against TLR1 (Cell Signalling Technology), TLR2 (D7G9Z, Cell Signalling Technology), MyD88 (D80F5, Cell Signalling Technology), TIRAP (EPR3509, Abcam), GFP (cross-reacts with Venus, 13.1/7.1, Roche), or Renilla luciferase (EPR17791, Abcam), followed by secondary probing with HRP-conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare) as previously described [44 (link)]. HRP-conjugated antibodies against β-actin (AC-15, Sigma) and GAPDH (Proteintech) were used to demonstrate even lane loading.

Twenty micrograms of total protein were resolved on 4–20% Tris HCl polyacrylamide gels using a Criterion electrophoresis system (BioRad; Hercules, CA, USA) at 200 V for 50 min at room temperature. The separated proteins were subjected to transfer onto nitrocellulose membranes (Thermo Fisher Scientific) by using a Criterion transfer system (Bio-Rad). The membranes were blocked in 5% nonfat dry milk 1× Tris-buffered saline (TBS) (Bio-Rad) (w/v) for 1 h at room temperature before being incubated in a 1:1000 dilution of primary antibody (anti-HSP70 (ab181606; Abcam, Waltham, MA, USA, annexin A2 (8235; Cell Signaling Tech, Danvers, MA, USA), GAPDH (649203; Biolegend, San Diego, CA, USA), syntenin (ab19903; Abcam) while rocking overnight at 4 °C. Thereafter, the membranes were washed three times with 1× TBS for 5 min intervals and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at a dilution of 1:3000 prepared in blocking solution while on a rocker for 1 h at room temperature. The membranes were then washed four times with 1× TBS for 4 min intervals, incubated with ECL reagent (BioRad) for 7 min at room temperature, and then developed (Bio-Rad imager).