Technical Reference #1757

1757.

Phospholipase D Promotes Lipid Microdomain-Associated Signaling Events in Mast Cells Felipe A. Lisboa, Ze Peng, Christian A. Combs, Michael A. Beaven, National Institutes of Health, Journal of Immunology, 183(1757), (2009)
Link To Paper

Materials & Methods:
Reagents were from the following sources: culture reagents from Life Technologies/Life Technologies; normal butyl alcohol (1-butanol) from Mallinckrodt; Tris from INC Biomedicals; mAb against Fc RI -subunit was provided by Dr. Juan Rivera (National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health); Abs against Fc RI -subunit, LAT, phospho-LAT Tyr226, and phosphotyrosine clone 4G10 from Millipore; Abs against Lyn, phospho-Src Tyr416, LAT, phospho- LAT Tyr171, phospho-LAT Tyr191 from Cell Signaling Technology; Abs against Thy-1, PLD1 and PLD2 from Santa Cruz Biotechnology; Ab against mouse Fc RI -subunit PE conjugated clone MAR-1 and isotype control from eBioscience; Vybrant Alexa Fluor 488 and 594 lipid raft labeling kits and NuPage LDS sample buffer 4 from Invitrogen; PLD1, PLD 2, and GAPDH plasmids from Integrated DNA Technologies; ONTARGETplus SMARTpool rat PLD1 and PLD2 small inhibitory RNA (siRNA) constructs from Dharmacon; pmaxGFP from Amaxa; and monoclonal anti-DNP IgE Ab (SPE-7), Ag (Ag, DNP-HSA, dinitrophenylated human serum albumin), and all other reagents from Sigma-Aldrich.

Microscopic Technique
Fluorescent optical and confocal laser microscopy

Cell Type(s)
RBL-2H3 cells