Technical Reference #1732

1732.

Lipid Rafts and Caveolin-1 Coordinate Interleukin-1B (IL-1B)-dependent Activation of NFkB by Controlling Endocytosis of Nox2 and IL-1B Receptor 1 from the Plasma Membrane Fredrick D. Oakley; Rachel L. Smith; John F. Engelhardt, University of Iowa, Journal of Biological Chemistry, 284(1732), (2009)
Link To Paper

Materials & Methods:
For the lipid raft inhibitor experiments assaying NF B activity we infected MCF-7 cells with an NF B luciferase reporter (Ad.NF B.luc) using recombinant adenovirus as described previously (25). Cells were used for experiments 48 h post-infection. The expression plasmids for FLAG-tagged IL-1R1 Nox2 GFP wild-type Vav1 (wtVav1) dominant-negative Vav1 (dnVav1) NF B luciferase reporter (NF B.luc) and GFP-caveolin-1 have been characterized previously (26–30). Transfection of the MCF-7 cells was performed by electroporation. Briefly cells were grown to 80% confluency on 150-mm plates. Cells were detached from the plates via trypsinization pelleted washed once with Opti-MEM medium and then resuspended at a concentration of 15 106 cells/800 l of Opti-MEM medium. The cells were mixed with the experimental plasmid (20 g/15 106 cells for the FLAG-IL-1R1 plasmid; 30 g/15 106 cells for the Nox2 GFP wtVav1 and dnVav1 plasmids; and 5 g/15 106 cells for theNF B.luc plasmid) and the mixture was placed in a Bio-Rad 0.4-cm gap cuvette. Electroporation was performed on a BTX ECM 830 electro-square porator with the following settings: voltage 225 V; pulse length 5.0 ms; number of pulses two; and interval between pulses 1.0 s. Following electroporation the cells were allowed to sit undisturbed for 10 min and then the contents of the cuvette were plated on a 150-mm cell culture dish with 20 ml of medium. The medium was changed at 24 h post-transfection and the cells were used for experiments at 48–72 h post-transfection.

Microscopic Technique
Confocal microscopy

Cell Type(s)
MCF-7 cells