Antibody against hsc 70

In vitro-differentiated BMDMs were pre-treated for 1 hour with 20 µM 13–197 or TPCA-1 in DMEM-10% FBS. Cells were then stimulated with TNFα (20 ng/ml) or LPS (100 ng/ml) at 37 °C in the presence of inhibitors for indicated period of time. At the end of stimulation, cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, and protease and phosphatase inhibitor cocktails). Equal amount of protein was separated on 8% SDS-polyacrylamide gels and transferred to PVDF membrane. Membranes were incubated with primary antibodies overnight at 4°C, washed, and incubated with secondary antibody conjugated with HRP for 1 hour. Membranes were developed by using Pierce ECL Western Blotting Substrate (Life Technologies, Grand Island, NY).
Antibodies against IκBα, phospho-IκBα, 4E-BP1, phospho-4E-BP1, S6K and phospho-S6K were from Cell Signaling Technology (Danvers, MA). Antibody against HSC70 was from Santa Cruz Biotechnology (Santa Cruz, CA).