Skip to main content
. 2015 Aug 23;98(6):963–974. doi: 10.1189/jlb.2VMA0515-229RR

Figure 4. Pellino-3 knockdown in 293/TLR4/MD2 cells enhances LPS-mediated activation of NF-κB and expression of CXCL-8 mRNA.

Figure 4.

293/TLR4 cells stably expressing scrambled, Pellino-3 shRNA (A–D) or Pellino-1 shRNA (A, B) were transiently transfected with pEFBOS-MD2 (A–D) alone or together with pELAM-Luc and pTK-Renilla-Luc (C). The cells were incubated for 3 h with medium (A), medium or 100 ng/ml LPS (B), or treated for 5 h (C) and 3 h (D) with medium, 10 or 100 ng/ml LPS (C, D). A, D) RNA was isolated, converted to cDNA, and subjected to RT-qPCR with primers specific for Pellino-1, Pellino-3, CXCL-8, and HPRT to examine expression of the depicted genes normalized to HPRT levels. B) Cell lysates were examined by Western blot analysis to determine the expression of Pellino-1 and Pellino-3 proteins relative to total p38 levels. (Right panels) Quantification of the immunoblots shown on left. C) Cell lysates were assayed for firefly vs. Renilla luciferase activities. Shown are the results of a representative experiment. Similar data were obtained in at least 3 other independent experiments. *P < 0.05.