Figure 7.

Biphasic phosphorylation of TRIM56 at Ser⁴⁷¹and Ser⁴⁷⁵and induction of IFNs following extracellular poly-I:C stimulation of HT1080-F55–derived cells expressing HA-Halo–tagged TRIM56.A, immunoblot analysis of the expression of HA-Halo–tagged TRIM56 or HA-Halo tags alone (using mouse anti-HA mAb) in HT1080-F55–derived cell clones stably transfected with HA-Halo-T56 (F55-HA-Halo-T56 clones) in comparison with cells expressing HA-Halo tags alone (F55-HA-Halo). Asterisk denotes a nonspecific band that served as a loading control. HA-Halo and HA-Halo tagged T56 are indicated using arrows. B, qRT-PCR analysis of the abundance of IL-29 (upper panel) or IFN-β (lower panel) transcript in F55-HA-Halo (dashed line) and F55-HA-Halo-T56 clone #2 (solid line) at indicated times post stimulation by 50 μg/ml poly-I:C. C, immunoblot analysis of the expression of the indicated proteins in F55-HA-Halo-T56 #2 cells at indicated time points post stimulation of 10 μg/ml poly-I:C. Cell lysates were immunoblotted (IB) with mouse anti-HA mAb (for total HA-Halo tagged T56), pAb specific for TRIM56 phosphorylated at Ser⁴⁷¹ or Ser⁴⁷⁵, rabbit anti-pSer³⁹⁸-IRF3, and rabbit anti-IRF-3 pAb (for total IRF3). Calreticulin serves as a loading control. IFN, interferon; IRF, interferon regulatory factor; TRIM56, tripartite-motif protein-56.