Cell lysate was prepared in radioimmunoprecipitation assay buffer lysis buffer
and debris completely removed by refrigerated centrifugation. The protein
concentration was determined by the bicinchoninic acid Protein Assay Kit
(ThermoFisher, MA, USA). An equal amount of protein was resolved by SDS-PAGE and
then transferred onto polyvinylidene difluoride (PVDF) membrane on ice. After
brief blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween-20
(TBST), the PVDF membrane was hybridized with indicated primary Abs
(anti-human-E-cadherin, 1:1000, #14472; anti-human-N-cadherin, 1:1000, #14215;
anti-human-vimentin, 1:1000, #3390; anti-mouse-IL-6, 1:1000, #12912;
anti-mouse-TNF-α, 1:1000, #11948; anti-GAPDH, 1:1000, #2118; Cell Signaling
Technology, MA, USA) at 4°C overnight. The unbound Abs were washed off with TBST
next day and incubated with specific secondary Abs (anti-rabbit IgG, HRP-linked
Ab, 1:5000, #7074; anti-mouse IgG, HRP-linked Ab, 1:5000, #7076; Cell Signaling
Technology, MA, USA) at room temperature (25°C) for 1 h. The PVDF membrane was
then rigorously washed with TBST for 30 min and target protein bands were
visualized using the Enhanced Chemiluminescence Kit (Millipore, UT, USA) in
accordance with the manufacturer’s instructions. The endogenous GAPDH was
employed as loading control.
and debris completely removed by refrigerated centrifugation. The protein
concentration was determined by the bicinchoninic acid Protein Assay Kit
(ThermoFisher, MA, USA). An equal amount of protein was resolved by SDS-PAGE and
then transferred onto polyvinylidene difluoride (PVDF) membrane on ice. After
brief blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween-20
(TBST), the PVDF membrane was hybridized with indicated primary Abs
(anti-human-E-cadherin, 1:1000, #14472; anti-human-N-cadherin, 1:1000, #14215;
anti-human-vimentin, 1:1000, #3390; anti-mouse-IL-6, 1:1000, #12912;
anti-mouse-TNF-α, 1:1000, #11948; anti-GAPDH, 1:1000, #2118; Cell Signaling
Technology, MA, USA) at 4°C overnight. The unbound Abs were washed off with TBST
next day and incubated with specific secondary Abs (anti-rabbit IgG, HRP-linked
Ab, 1:5000, #7074; anti-mouse IgG, HRP-linked Ab, 1:5000, #7076; Cell Signaling
Technology, MA, USA) at room temperature (25°C) for 1 h. The PVDF membrane was
then rigorously washed with TBST for 30 min and target protein bands were
visualized using the Enhanced Chemiluminescence Kit (Millipore, UT, USA) in
accordance with the manufacturer’s instructions. The endogenous GAPDH was
employed as loading control.