Transfection of Jurkat, HEK 293T, and Primary T Cells

Jurkat T cells were transfected as previously described using the ECM 830 Square Wave Electroporation system (BTX) (32 (link)). The following SMARTpool ON-TARGET Plus siRNA duplexes were purchased from Thermo Scientific: non-targeting pool (D-001810-10-05) and human Elmo1 (L-012851-00-0005). HEK 293T cells were transfected with 1μg empty pEBB-Flag (vector) or Dock2-Flag (from M. Matsuda(5 (link))) plus 4μg pEBB-Elmo-Flag plasmids by calcium phosphate (Profection, Promega). Primary T cells were transfected using the Mouse T cell Nucleofector Kit (Amaxa), with 2×10⁶ CD4+ T cells, 1μg pMAX-GFP (Amaxa) and 4μg expression vectors. Elmo expression plasmids the pEBB-Flag backbone were provided by K.S. Ravichandran. MT123-HA-ubiquitin vector provided by Dirk Bohmann(33 (link)).

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Stevenson C., de la Rosa G., Anderson C.S., Murphy P.S., Capece T, & Elliott M.K. (2014). Essential role of Elmo1 in Dock2-dependent lymphocyte migration. Journal of immunology (Baltimore, Md. : 1950), 192(12), 6062-6070.

Publication 2014

Profection Mouse T cell Nucleofector Kit pMAX-GFP

293t cells Backbone Calcium phosphate Cd4 t cells Electroporation Human Jurkat cells Mouse Plasmids Promega Sirna T cell Ubiquitin Vector

Corresponding Organization : University of Rochester Medical Center

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Variable analysis

independent variables

Transfection method
SiRNA duplexes
Plasmid constructs

dependent variables

Elmo1 expression
Dock2 expression
Ubiquitin expression

control variables

Cell lines (Jurkat T cells, HEK 293T cells, Primary T cells)
Electroporation system (ECM 830 Square Wave Electroporation system)
Transfection methods (calcium phosphate, Nucleofector Kit)

controls

Negative control: non-targeting siRNA pool
Positive control: not explicitly mentioned

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