Fig. 5.

PLCβ2 inhibits TAK1 activation via PIP2. a, b Reporter assay of NF-κB (b) and AP-1 (c) activation in HEK293T cells expressing TAK1 and TAB1 in the presence or absence of PLCβ2 (WT) or phospholipase-inactive mutant (M). c IB of cell lysates from HEK293 cells expressing TAK1 and TAB1 in the presence or absence of PLCβ2 (WT) or phospholipase-inactive mutant (M). d–g IB of cell lysates from macrophages treated with PIP2 in a lipid carrier for 10 min or 10 mM neomycin for 2 h before stimulating with poly(I:C) for the indicated times using anti-phospho-antibodies. h, i Q-PCR analysis of relative Tnf (h) and Il6 (i) mRNA levels in macrophages pretreated with neomycin before stimulating with poly(I:C) for the indicated time. j IB of cell lysates from wild-type or Plcb2^−/− macrophages treated with 10 mM neomycin for 2 h before stimulating with poly(I:C) for the indicated times using phospho-antibodies. k, l Q-PCR analysis of relative Tnf (k) and Il6 (l) mRNA levels in wild-type or Plcb2^−/− macrophages pretreated with 10 mM neomycin for 2 h before stimulating with poly(I:C) for 6 h. m Phospholipid-binding assays with purified FLAG-TAK1 protein from HEK293 cells. PI phosphatidyl- inositol, TG triglyceride, DAG diacylglycerol, PA phosphatidic acid. n Dotting assay of PI(4,5)P2 with purified TAK1 and mutants. o, p TAK1^−/− A549 cells complemented with wild-type TAK1 or its W241A or N245A mutants and stimulated with poly(I:C) for the indicated times before assay via immunoblotting with anti-phospho-antibodies (o) or Q-PCR analysis of relative Il6 mRNA (p). ns not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.001 by unpaired t-test (a, b, h, i, k, l and p). Data are representative of three experiments with at least three independent biological replicates (mean and s.e.m. of n = 3 cultures in d–l)