Fig. 5: SPOP is specifically involved in the TLR-MyD88-mediated signaling.

(a) Effects of SPOP on TLR2 signaling. RAW264.7 cells (4 × 10⁵) were transfected with the indicated siRNA (2 μg each) for forty hours. Then the cells were treated with PGN (50 μg/mL) for 2 hours and the total RNA was prepared for qPCR analysis. (b) Effects of SPOP on TLR3 signaling. The experiments were similarly performed as in (a), except that the stimuli is Poly(I:C) (50 μg/mL). (c) Effects of SPOP on TLR7 signaling. The experiments were similarly performed as in (a), except that the stimuli is R848 (40 nM). (d) Effects of SPOP on TLR9 signaling. The experiments were similarly performed as in (a), except that the stimuli is CpG (2 μM). (e) Knockdown of SPOP increased aggregation of MyD88 induced by PGN. RAW264.7 cells (2 × 10⁶) were transfected with a control siRNA or SPOP-siRNA#2 (10 μg each) for forty hours, then the cells were treated with PGN (50 μg/mL) for the indicated times. Cell lysates were separated by native- or SDS-PAGE as indicated and analyzed by immunoblots with the indicated antibodies. (f) Knockdown of SPOP increased aggregation of MyD88 induced by R848. The experiments were similarly performed as in (e), except that the stimuli is R848 (40 nM). (g) Knockdown of SPOP increased aggregation of MyD88 induced by CpG. The experiments were similarly performed as in (e), except that the stimuli is CpG (2 μM). Graphs show mean ± SD, n = 3. *p < 0.05; **p < 0.01.