Fig 4. UL138-USP1 regulates pSTAT1 independently of TBK1 stabilization.
(A) Fibroblasts were reverse transfected with a siRNA against TBK1 for 48 hours prior to being infected (MOI = 1) with a WT or ΔUL138STOP virus. Lysates were collected and immunoblotted for pSTAT1, STAT1, TBK1, IE1/2, and Tubulin. (B) Quantification of TBK1 levels normalized to NTC and graphed for statistical analysis with an unpaired t test and represented by asterisks; ***p-value <0.001. (C) Fibroblasts were infected (MOI = 1) with either WT or ΔUL138STOP virus and lysates were immunoblotted. pTBK1, TBK1, pSTING, STING, IE1/2, and Tubulin were detected using antibodies. (D) Fibroblasts were treated with 0.88 μM C527 for 24 hours prior to being infected (MOI = 1) with a WT or ΔUL138STOP virus. Lysates were collected and immunoblotted for TBK1 and tubulin. (E) Fibroblasts were infected (MOI = 1) with a WT or ΔUL138STOP virus and lysates were collected and immunoblotted for pTYK2, TYK2, pJAK1, JAK1, pSTAT1, STAT1, IE1/2, and Tubulin. (F) Fibroblasts were infected (MOI = 1) with a WT or ΔUL138STOP virus and lysates were collected and immunoblotted for pSTAT1, STAT1, pSHP-2, SHP-2, PTPN2, IE1/2, and Tubulin. (G) Fibroblasts were infected with a WT or ΔUL138STOP virus (MOI = 1) for 48 hours before treatment with DMSO (vehicle control) or 10 μM of MG132 for 6 hours before lysates were collected. Lysates were immunoblotted for pSTAT1, STAT1, IE1/2, and Tubulin using antibodies. (H) The fold increase in rescue in the MG132 treated cells over the DMSO treated for mock, WT, and ΔUL138STOP infected cells were graphed.