Skip to main content
. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Nat Immunol. 2011 Mar 13;12(4):335–343. doi: 10.1038/ni.2007

Figure 5. Phosphorylation of RPS3 at serine 209 is critical for its nuclear translocation and NF-κB specifier function.

Figure 5

(a) Immunoblotting of the cytosolic (Cyto) and nuclear (Nuc) subcellular fractions derived from Jurkat cells overexpressing wild-type or S209A mutant Flag-RPS3 and stimulated with PMA plus ionomycin (PMA+I). Hsp90 and PARP were the cytoplasmic and nuclear markers, respectively (left). Densitometry of all bands was performed, and the intensity of each RPS3 band was normalized to loading hsp90 or PARP controls. The percentage of relative RPS3 was further normalized to the 0-min samples (set as 100%) in cells stimulated with or without PMA+I (right). (b) Immunoblotting of the cytosolic (Cyto) and nuclear (Nuc) subcellular fractions derived from Jurkat cells overexpressing IKKβ together with wild-type or S209A mutant Flag-RPS3 (left). Densitometry of all bands was performed and normalized as in (a), except further normalized to the control samples without overexpressing IKKβ (right). (c) Immunoblotting for endogenous and ectopically expressed RPS3 proteins in whole-cell lysates from Jurkat cells transfected with siRNA specifically targeting the 3′ untranslated region of RPS3 mRNA (RPS3-3′ UTR) or scrambled nonspecific siRNA (NS), plus wild-type (WT) or S209A mutant (S209A) Flag-RPS3 constructs. β-actin serves as a loading control. (d) NF-κB luciferase assay (mean and s.d., n = 3) of Jurkat cells transfected with siRNA specifically targeting the 3′ untranslated region of RPS3 mRNA (RPS3-3′ UTR) or scrambled nonspecific (NS) siRNA, together with either wild-type (WT) or S209A mutant (S209A) Flag-RPS3 constructs. A 5 × Ig κB site-driven luciferase construct was used as the reporter gene. Not statistically significant (NS), ** P < 0.01, calculated by Student's t-test. (e) Jurkat cells were left untreated (-) or stimulated (+) with PMA plus ionomycin (PMA+I) for 1 h, after transfection with siRNAs and Flag-RPS3 constructs as in (d). Cell extracts were analyzed by chromatin immunoprecipitation assays for the recruitment of Flag-RPS3 and endogenous p65 proteins to the promoters of NFKBIA, IL8, or ACTB, using Flag and p65 antibodies, respectively. Flag-RPS3- and p65-bound DNA was analyzed by quantitative real-time PCR in triplicate (primers, above diagrams) and normalized to input DNA, compared to cells transfected with a WT Flag-RPS3 construct and left untreated. Data are representative of at least of two independent experiments.