The cells were trypsinized, harvested, and lysed, and they were then subjected to protein
extraction. The obtained total proteins were electrophoresed on SDS-PAGE gels and
subsequently transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Skim milk
powder was diluted in TTBS solution. After that, the membranes were blocked with diluted
milk and incubated with primary antibodies against α-ENaC (Proteintech Group, Wuhan,
China; 1:500 dilution), β-ENaC (Proteintech Group; 1:500 dilution), γ-ENaC (Proteintech
Group; 1:1,000 dilution), ERK (Proteintech Group; 1:500 dilution), p-ERK (BIOSS, Beijing,
China; 1:400 dilution), p38 (BIOSS; 1:400 dilution), p-p38 (BIOSS; 1:400 dilution), JNK
(Proteintech Group; 1:1,000 dilution) and p-JNK (Abcam, Cambridge Science Park, Cambridge,
UK ; 1:1,000 dilution) at 4°C overnight. HRP-conjugated secondary antibody (Beyotime
Institute of Biotechnology; 1:5,000 dilution) was added after washing with TTBS. Bands
were subsequently visualized using ECL reagent (Beyotime Institute of Biotechnology). The
optical densities of bands were analyzed by Gel-Pro Analyzer (Media Cybernetics, Inc.,
Bethesda, MD, USA).
extraction. The obtained total proteins were electrophoresed on SDS-PAGE gels and
subsequently transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Skim milk
powder was diluted in TTBS solution. After that, the membranes were blocked with diluted
milk and incubated with primary antibodies against α-ENaC (Proteintech Group, Wuhan,
China; 1:500 dilution), β-ENaC (Proteintech Group; 1:500 dilution), γ-ENaC (Proteintech
Group; 1:1,000 dilution), ERK (Proteintech Group; 1:500 dilution), p-ERK (BIOSS, Beijing,
China; 1:400 dilution), p38 (BIOSS; 1:400 dilution), p-p38 (BIOSS; 1:400 dilution), JNK
(Proteintech Group; 1:1,000 dilution) and p-JNK (Abcam, Cambridge Science Park, Cambridge,
UK ; 1:1,000 dilution) at 4°C overnight. HRP-conjugated secondary antibody (Beyotime
Institute of Biotechnology; 1:5,000 dilution) was added after washing with TTBS. Bands
were subsequently visualized using ECL reagent (Beyotime Institute of Biotechnology). The
optical densities of bands were analyzed by Gel-Pro Analyzer (Media Cybernetics, Inc.,
Bethesda, MD, USA).