Fig. 6: UDPG-P2Y₁₄ pathway regulates STAT1 phosphorylation.

a, b BMDMs were pretreated with GPI (20 or 50 μM) for 30 min or pretransfected with Pygl siRNA for 24 h prior to stimulation with IFN-γ/LPS, followed by western blot analysis of STAT1 and its phosphorylation from 15 to 120 min after stimulation. c, d Pygl siRNA transfected BMDMs were treated with IFN-γ/LPS ± UDPG, STAT1 and its phosphorylation was analyzed by two-photon confocal microscope, scale bar, 10 μm (c) and western blot (d). e BMDMs were pretransfected with Pygl siRNA for 24 h prior to stimulation with IFN-γ/LPS, followed by western blot analysis of JAK1 and JAK2 from 15 to 120 min after stimulation. f, g BMDMs were pretreated with GPI alone or combined with MG132 (f) or Na₃VO₄ (g) for 30 min prior to stimulation with IFN-γ/LPS, followed by western blot analysis of STAT1, JAK1, and JAK2 from 15 to 120 min after stimulation. h BMDMs were pretreated with GPI for 30 min prior to stimulation with IFN-γ/LPS, the cytoplasmic and nuclear protein fractions were blotted for TC45, β-actin (cytoplasmic marker) and Histone H3 (nuclear marker). i Ptpn2 siRNA transfected BMDMs were stimulated with IFN-γ/LPS, STAT1, JAK1, JAK2, and TC45 were analyzed by western blot. j Pygl siRNA transfected BMDMs were stimulated with IFN-γ/LPS ± UDPG, ERK, JNK, P38, JAK1, JAK2, and TC45 were analyzed from 15 to 120 min after stimulation by western blot. k, l IFN-γ/LPS stimulated BMDMs were treated with UDPG alone or combined with U0126, SP600125 or SB203580 for 1 and 3 h, JAK1, JAK2, STAT1, and TC45 were analyzed by western blot (k). Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (l). Data are presented as mean ± SEM of n = 3 biologically independent experiments. P values were calculated using one-way ANOVA.