Protein extracts were separated on 10%
sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose
membranes. Membranes were blocked with 5% fat-free milk at room temperature
for 1 h and then incubated overnight at 4 °C with a primary antibody
(Ab), followed by incubation with the appropriate horseradish-peroxidase-conjugated
reporter Ab (eBioscience, San Diego, CA) for 1 h at room temperature.
The enhanced chemiluminescence FluorChem E system (Bio-Techne, Minneapolis,
MN) was used for developing the reaction. Abs specific for the total
and phosphorylated forms of ERK1/2 (Thr202/Tyr204), JNK1/2 (Thr183/Tyr185),
and p38 (Thr180/Tyr182) were obtained from Cell Signaling Technology
(Danvers, MA). An Ab against β-actin was purchased from Santa
Cruz Biotechnology (Dallas, TX).
sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose
membranes. Membranes were blocked with 5% fat-free milk at room temperature
for 1 h and then incubated overnight at 4 °C with a primary antibody
(Ab), followed by incubation with the appropriate horseradish-peroxidase-conjugated
reporter Ab (eBioscience, San Diego, CA) for 1 h at room temperature.
The enhanced chemiluminescence FluorChem E system (Bio-Techne, Minneapolis,
MN) was used for developing the reaction. Abs specific for the total
and phosphorylated forms of ERK1/2 (Thr202/Tyr204), JNK1/2 (Thr183/Tyr185),
and p38 (Thr180/Tyr182) were obtained from Cell Signaling Technology
(Danvers, MA). An Ab against β-actin was purchased from Santa
Cruz Biotechnology (Dallas, TX).