Bel-7404 and HepG2 cells were plated in 60-mm plated and grown to 70%
confluence. Cells were treated as indicated, washed with cold PBS, and lysed
using standard procedures. Cell lysates were resolved by SDS/PAGE and
transferred on to a PVDF membrane. The membrane was blocked with 5% skim
milk (m/v) in Tris-buffered saline with Tween-20 (TBST; 50 mM
Tris/HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) at room temperature
for 1 h and incubated with IL-6R, gp130, STAT3, p-STAT3, or tubulin antibodies
(Cell Signaling Technology, Beverly, MA, U.S.A.) at 4°C overnight. The
membrane was then washed and incubated with horseradish peroxidase
(HRP)–conjugated secondary antibodies (Cell Signaling Technology)
following exposure to Immobilon™ Western Chemiluminescent HRP Substrate
(Millipore, New Orleans, LA, U.S.A.).
confluence. Cells were treated as indicated, washed with cold PBS, and lysed
using standard procedures. Cell lysates were resolved by SDS/PAGE and
transferred on to a PVDF membrane. The membrane was blocked with 5% skim
milk (m/v) in Tris-buffered saline with Tween-20 (TBST; 50 mM
Tris/HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) at room temperature
for 1 h and incubated with IL-6R, gp130, STAT3, p-STAT3, or tubulin antibodies
(Cell Signaling Technology, Beverly, MA, U.S.A.) at 4°C overnight. The
membrane was then washed and incubated with horseradish peroxidase
(HRP)–conjugated secondary antibodies (Cell Signaling Technology)
following exposure to Immobilon™ Western Chemiluminescent HRP Substrate
(Millipore, New Orleans, LA, U.S.A.).