Phospho jak3

Homogenized liver and spleen tissues, and splenocytes from spleen of WT mice and Jurkat T cells lysed by protein extraction solution (PRO-PREP, iNtRON Biotechnology) containing protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Total proteins (30 μg) were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk overnight and then incubated with primary antibodies (diluted 1 : 1000) for 1 h at room temperature. The membranes were immunoblotted with following primary antibodies: Phospho-STAT1, Phospho-JAK3, Phospho-JNK, Phospho-PLCγ, Phospho-IκB (Cell Signaling Technology, Beverly, MA, USA), STAT1, JAK3, JNK, PLCγ, IκB (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with Tris-buffered saline containing 0.05% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1 : 3000) for 1 h at room temperature. Binding of antibodies to the PVDF membrane was detected with enhanced chemiluminescence solution (Amersham Bioscience, Buckinghamshire, UK) and X-ray film (AGFA, Mortsel, Belgium).