Fig. 5
NLGP-Dectin-1 interaction transduces a PKCδ-mediated intracellular signal to NFκB subunits through CARD9-Bcl10-MALT1-based signaling axis. a Quantitative PCRs measuring IL-10 and IL-12A or IL-12p35 transcripts on NLGP treatment to tumor-conditioned mBMDCs and effects of inhibiting NLGP-Dectin-1 interactions subsequently by Laminarin, RNAi-based Dectin-1 depletion and both. β actin was taken as endogenous control, fold changes were deduced by Livak method (2.−ΔΔCt) from replicates (n = 3) (data are represented as mean ± SD). b General interactions among probable second messengers of NLGP-Dectin-1 association-induced signals in DCs, deduced from STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) (Ver. 11) based on their reported physical (physical network) (right) and distal (full network) (left) associations. The PrkCD node stands for Protein kinase C δ (PKCδ). c Western blotting to measure PKCδ and phospho-PKCδ (pPKCδ) in same experimental mBMDC groups as (a). β actin was kept as loading control (n = 3) (data from replicates are represented as mean ± SD). d Western blots to quantify levels of Bcl10 and MALT1 (both bound to CARD9) in these groups by co-immunoprecipitation, as CARD9 was pulled with its antibody out of total cell protein lysates. Levels of CARD9 and all three proteins (within total protein lysates) were kept as positive control (upper left). Data from replicates (n = 3) are represented as mean ± SD (upper right, lower left and lower right). e Western blots, measuring p65 (also known as RelA from the Rela gene) and p50 (of NFKB1, transcribed from the Nfkb1 gene) proteins within cytosol and nuclei in these mBMDC groups. β actin and Histone H3 were taken as loading controls for cytosolic and nuclear proteins respectively. Data from replicates (n = 3) are represented as mean ± SD (middle and right). Numerical data of (a) were tested by one-way ANOVA (F = 126.2 and 29.19, df = 23 and 23) followed by Tukey’s multiple comparisons test, and those of Figures (c), (d) and (e) were tested by two-way ANOVA followed by Tukey’s multiple comparisons test. Normal distribution of the data were verified before each test (See also Fig. S2e-l)