Fig 1: Protein kinase D promotes Sp1 activity over NF-kB or p38. 16HBE cells were pre-treated with 5 µM CRT for 2 h prior to stimulation with 5 µg/ml polyI:C as indicated for 20 mins. Whole cell lysates were analyzed for phospho-p65/NF-kB (A) and phospho-p38/MAPK (B) by Western blot. (C) 16HBE cells were transfected with siRNA as in Figure 4 prior stimulation with 5 µg/ml polyI:C for 15 mins and whole cell extracts analyzed by Western. Three independent wells treated and analyzed for each condition (replicates denoted by “1, 2, 3”). (D) PKD3 was knocked down in 16HBE cells with 20 pmol siRNA overnight. Cells were then treated with 5 µg/ml polyI:C for 15min and nuclear extracts analyzed for activation of a panel of transcription factors using an unbiased DNA-binding assay kit (TF Profiling Plate Array 1 Kit, Signosis, see Methods). The top 5 transcription factors induced by polyI:C and inhibited by PKD3 siRNA knock-down are shown. See Supplemental Table 1 for complete list. (E) 16HBE cells were pretreated 2h with 5 µM CRT before stimulation with polyI:C (5 µg/ml) for 5 min. Nuclear extract was harvested and Sp1 activity analyzed via DNA binding assay (RayBiotech). Results of two independent experiments are shown. *** indicates p<0.001 determined using one way ANOVA followed by Tukey’s t-test.