Skip to main content
. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Immunol. 2011 Jan 26;186(5):2959–2969. doi: 10.4049/jimmunol.1000112

FIGURE 3. Triad3A suppresses 2DL4-mediated production of IL-8 in 293T cells.

FIGURE 3

A, Triad3A co-expression suppresses 2DL4-mediated IL-8 production. 293T cells were transfected with FLAG-2DL4 ± FLAG-Triad3A cDNAs and tested 48 h later for IL-8 secretion by ELISA. Results are mean ±S.D. of single assays from 8 experiments. B, Triad3A knockdown reduces 2DL4 ubiquitylation in 293T cells. 293T cells were transfected with cDNAs encoding combinations of FLAG-2DL4, FLAG-Triad3A, HA-Ub, and one of three shRNAs targeting Triad3A. After 48 h, cells were lysed and 2DL4 immunoprecipitates (top panel) or cell lysates (bottom panels) were immunoblotted as indicated. C, 2DL4-mediated IL-8 production was restored by Triad3A knockdown. 293T cells were transfected with indicated combinations of FLAG-2DL4, FLAG-Triad3A, and one of the shRNA constructs, and tested 48 h later for IL-8 production by ELISA. Results are mean ±S.D. of single determinations from 3 experiments. D, Knockdown of endogenous Triad3A significantly enhanced 2DL4-mediated IL-8 production in 293T cells. 293T cells were transfected with shRNA3 and stably transfected cells were selected by treating with puromycin (2 μg/ml) for 7 days. The shRNA transfectants and 293T parent cells were then transfected with 2DL4 and assayed 48 hrs later for IL-8 secretion by ELISA (top panel). Results are mean ±S.D. of a single assay from 3 experiments. Lysates from 293T transfectants were immunoblotted with anti-Triad3A and anti-GAPDH to assess knockdown efficiency (bottom). Note that 10-fold less lysate was added for the Triad3A transfected sample (lane 3).