Sds polyacrylamide gel

The hTERT-transfected HNECs were scraped from a 60 mm dish containing 300 μl of buffer (1 mM NaHCO3 and 2 mM phenylmethylsulfonyl fluoride), collected in microcentrifuge tubes, and then sonicated for 10 s. The protein concentrations of the samples were determined using a BCA protein assay reagent kit (Pierce Chemical Co.; Rockford, IL). Aliquots of 15 μl of protein/lane for each sample were separated by electrophoresis in 5–20% SDS polyacrylamide gels (Wako, Osaka, Japan), and electrophoretically transferred to a nitrocellulose membrane (Immobilon; Millipore Co.; Bedford, UK). The membrane was saturated for 30 min at room temperature with blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20, and 4% skim milk) and incubated with anti-CK5, anti-CK7, anti-p63, anti-ΔNp63, anti-anti-RSV-G protein^{21 (link)}, anti-occludin, anti-CLDN-1, -4, -7, anti-LSR, anti-tricellulin, anti-Ac-tubulin, anti-γ-tubulin, anti-phospho-NFκB, anti-NFκB, anti-phospho-p38 MAPK and anti-p38 MAPK antibodies (1:1000) at room temperature for 1 h. Then it was incubated with HRP-conjugated anti-mouse and anti-rabbit IgG antibodies at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blotting system.

Cultured cells were scraped from a 35 mm dish containing 400 μL of buffer (1 mM NaHCO₃ and 2 mM phenylmethylsulfonyl fluoride), and HSDE cells were scraped from a 60 mm dish containing 300 μL of buffer, collected in microcentrifuge tubes, and then sonicated for 10 s. The protein concentrations of the samples were determined using a BCA protein assay reagent kit (Pierce Chemical Co.; Rockford, IL, USA). Aliquots of 15 μL of protein/lane for each sample were separated by electrophoresis in 5–20% SDS polyacrylamide gels (Wako, Osaka, Japan), before being electrophoretically transferred to a nitrocellulose membrane (Immobilon; Millipore Co.; Bedford, UK). The membrane was saturated for 30 min at room temperature with blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween-20, and 4% skim milk), and incubated with anti-p63, anti-CGN, anti-ZO-3, anti-OLFML3, anti-CYPLA1, anti-LSR, anti-TRIC, anti-CLDN-1, -4, -7, anti-acetylated-tubulin, anti-pMAPK, and anti-actin antibodies (1:1000) at room temperature for 1 h or overnight. It was then was incubated with HRP-conjugated anti-mouse and anti-rabbit IgG antibodies at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blotting system.

The cultured cells were scraped from 60 mm dishes containing 400 μl of buffer (1 mM NaHCO³ and 2 mM phenylmethylsulfonyl fluoride), collected in microcentrifuge tubes, and then sonicated for 10s. The protein concentrations of the samples were determined using a BCA protein assay regent kit (Pierce Chemical Co.; Rockford, IL, USA). Aliquots of 15 μl of 20 μg protein/lane for each sample were separated by electrophoresis in 5–20% SDS polyacrylamide gels (Wako, Osaka, Japan), and electrophoretically transfered to a nitrocellulose membrane (Immobilon; Millipore Co.; Bedford, UK). The membrane was saturated with blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20, and 4% skim milk) for 30 min at room temperature and incubated with anti-LSR, anti-TRIC, anti-CLDN-1, -2, anti-Ac-tubulin and anti-actin antibodies (1:1000) at room temperature overnight. Then it was incubated with HRP-conjugated anti-mouse and anti-rabbit IgG antibodies at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blotting system.