Gene Silencing with siRNA Depletion

Gene silencing with siRNA was performed as described (23 (link), 30 (link), 49 (link)). In brief, to deplete α_1B/D-ARs from the cell surface, cells were incubated in Nunc six-well plates (Thermo Fisher Scientific) for 3 d in Accell transfection media (GE Dharmacon) with α_1B-AR, α_1D-AR, or NT (negative control) siRNA (GE Dharmacon) at a concentration of 1 µM. On day 3, cells were centrifuged at 300 × g for 5 min and resuspended in RPMI 1640 (Sigma) supplemented with 10% HyClone FBS from Cytiva (Marlborough, MA), 100 μg/mL penicillin from Invitrogen (Waltham, MA), and 100 μg/mL streptomycin (Invitrogen). Cells were utilized on day 4 for experimentation.

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Enten G.A., Gao X., Strzelinski H.R., Weche M., Liggett S.B, & Majetschak M. (2022). α1B/D-adrenoceptors regulate chemokine receptor–mediated leukocyte migration via formation of heteromeric receptor complexes. Proceedings of the National Academy of Sciences of the United States of America, 119(20), e2123511119.

Publication 2022

Nunc six-well plates Accell transfection media RPMI 1640 HyClone FBS penicillin streptomycin

Cells Penicillin Sirna Streptomycin Transfection

Corresponding Organization :

Other organizations : University of South Florida

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Variable analysis

independent variables

α1B-AR siRNA
α1D-AR siRNA
NT (negative control) siRNA

dependent variables

α1B/D-AR depletion from the cell surface

control variables

Cells incubated in Nunc six-well plates
Cells incubated in Accell transfection media
Cells incubated for 3 days
Cells centrifuged at 300 × g for 5 min
Cells resuspended in RPMI 1640 supplemented with 10% HyClone FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin
Cells utilized on day 4 for experimentation

controls

Positive control: α1B-AR siRNA, α1D-AR siRNA
Negative control: NT (negative control) siRNA

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