Fig. 3.

Fyn–PI3K–PKCα axis inactivates SHP-1-mediated ITAMi signaling. a Modulation of LPS-mediated IL-8 production by Lyn or Fyn after induction of FcγRIIA-ITAM signal on transfected THP-1. Cells were stimulated with LPS for 1 h at 37 °C after induction of ITAM as described in Fig. 1a. Data are presented as the mean ± s.e.m. ***P < .001; Student’s unpaired t-test. b After induction of FcγRIIA-mediated ITAMi or ITAM signals on BMDM derived from FcγRIIA transgenic mice or from FcγRIIA^Tg under Lyn-deficient or Fyn-deficient backgrounds. Cell lysate samples were subjected to SDS-PAGE and immunoblots were performed using anti-phospho (p) SHP-1 serine 591 (S591) or tyrosine 536 (Y536) Abs. c Effect of PI3K, PKC, and ERK inhibitors on SHP-1 phosphorylation driven by ITAM signals in BMDMs. Cells were pre-treated with inhibitors as indicated and Western blotting was performed on cell lysates using antibodies anti-phosphorylated (p) kinases and phosphatases as indicated. Total protein contents in cell lysates were shown in Supplementary Fig. 3,b. d–f Involvement of PKCα on serine SHP-1 phosphorylation following FcγRIIA-ITAM signal. BMDMs from FcγRIIA^Tg (d), Lyn-deficient FcγRIIA^Tg (e), or Fyn-deficient FcγRIIA^Tg (f) mice were transfected with indicated siRNAs before induction of FcγRIIA-ITAM as described in Fig. 1a. b–f Are representative of three experiments. NS, not stimulated