Fig. 6

Poly (I:C) increased phosphorylation of TLR3 tyrosine residue and following recruitment of PI3K. a HEK 293 cells expressing Myc-tagged TLR3 and treated with poly(I:C). Cell lysates were immunoprecipitated with anti-phosphotyrosine (PY20) and western blotted with anti-TLR3. b Cell lysates were immunoprecipitated with anti-Myc and western blotted with anti-PI3K-p85-subunit. The same blot was reprobed with the TLR3 antibody as an immunoprecipitation control. c Mice were injected with poly(I:C), and heart tissues were harvested at 0, 15, 30, 120, and 240 min. Samples were immunoprecipitated with antiphosphotyrosine (PY20) and western blotted with anti-TLR3 (upper). Furthermore, samples were immunoprecipitated with anti-TLR3 and western blotted with anti-PI3K. The same blot was reprobed with the TLR3 antibody as immunoprecipitation control (below). d Mice were pretreated with or without poly(I:C) and subjected to I/R. Twenty-four hour after I/R heart tissues were taken, and samples were immunoprecipitated with anti-TLR3 and western blotted with anti-PI3K. The same blot was reprobed with TLR3 antibody as an immunoprecipitation control. e, f Representative photograph (e) and quantification (f) of PLA reaction between TLR3 and PI3K were shown (n = 6) (all experiment groups were compared via one-way ANOVA with a Turkey’s multiple comparisons test, bars indicate the SEM, *P < 0.05; **P < 0.01). Scale bar (first line), 100 μm; scale bar (second line), 50 μm; scale bar (third line), 10 μm. g The schematic figure revealed poly(I:C) protects the hearts against I/R injury through phosphorylation of TLR3 tyrosine residue, activation of PI3K and recruitment of Akt, resulting in decreased of NF-κB activity, and reduced inflammatory and apoptotic responses