Technical Reference #1797

1797.

Glucocorticoid-mediated Inhibition of Lck Modulates the Pattern of T Cell Receptor-induced Calcium Signals by Down-regulating Inositol 1;4;5-Trishosphate Receptors Michael W. Harr; Yiping Rong; Martin D. Bootman; H. Llewelyn Roderick; Clark W. Distelhorst, Case Western Reserve University University Hospitals of Cleveland Babraham Institute University of Cambridge, Journal of Biological Chemistry, 284(1797), (2009)
Link To Paper

Materials & Methods:
Fura-2AM was purchased from Invitrogen. Dasatinib (BMS-354825 Sprycel) and thapsigargin were purchased from LC laboratories (Woburn MA). Dexamethasone was purchased from Sigma. Lck and IP3 receptor siRNAs were purchased from Dharmacon (Lafayette CO). The following antibodies were used in this study: anti-mouse CD3e (145-2C11) and IP3 receptor 3 (BD Biosciences); Lck (3A5) (Santa Cruz Biotechnology Santa Cruz CA); phospho-Lck Tyr-394 (Src Tyr-416 2101) (Cell Signaling Technology Danvers MA); -actin (AC-15) (Sigma). Antibody against Type I IP3 receptor was kindly provided by Jan Parys and HumbertDeSmedt (Rbt 03) (KU Leuven Belgium) (26). Antibodies for Type I and Type II IP3 receptors were also kindly provided by Richard Wojcikiewicz (CT-1 and CT-2; SUNY Syracuse NY) (27 28).

Microscopic Technique
inverted Zeiss axiovert S100 microscope and 20X Fluor objective

Cell Type(s)
WEHI7.2 cells