ZCCHC3 protein associates with MOV10 and ZAP proteins.

(A) Immunoprecipitated FLAG-tagged ZCCHC3 protein associates with both exogenously expressed (top panels) and endogenous (bottom panels) MOV10 protein in an RNA-dependent manner (last two lanes, -/+ RNaseA treatment). Control vectors were pcDNA5 FRT/TO (top panels) and pcDNA6 myc/his B (bottom panels). (B) α-ZCCHC3-CS antibody bound to Protein G Dynabeads was used in two separate IP experiments [1 and 2] to show that immunoprecipitated endogenous ZCCHC3 protein binds endogenous MOV10 protein and that this interaction is lost with RNaseA treatment (lanes 6 and 7). Normal rabbit IgG was used as negative control and showed no binding of endogenous MOV10 protein (last 2 lanes). (C) FLAG-tagged ZCCHC3 IPs endogenous ZAP protein in an RNA-independent manner (last two lanes, -/+ RNaseA treatment). To generate this data, the Western blot of Fig 6A (lower panel) was stripped. (D) FLAG-tagged ZAP-L long isoform (PARP13.1) co-IPs V5-TEV-ZCCHC3 protein in an RNA-independent manner (last two lanes, -/+). Experiments in A-D were performed in 293T cells. (E and F) Tagged V5-TEV-ZCCHC3 colocalizes with endogenous MOV10 and ZAP proteins in cells stressed with Na-arsenite. (G) Exogenously expressed ZCCHC3-FL and V5-TEV-TRIM25 proteins colocalize in cytoplasmic granules of stressed U2OS-WT cells. (H) Multiple 293T cell lines deleted for ZAP expression by CRISPR protocol (in red text, KO cell lines AE-BB-B8-P3 and AE-CC-D8-P9 are shown as examples) have diminished expression of endogenous ZCCHC3 compared with control lines (top panels). Control lines (ex., AB-B-B5-P5 and AE-CC-E4-P9) were similarly subjected to CRISPR treatment but without successful KO of ZAP expression. Full-length (ZAP-L) and the short (ZAP-S) isoforms of ZAP are indicated by arrows on the Western blot. Reprobing with α-HSP90 antibody and staining of Western transfer blots with Ponceau S (bottom panels) are controls for equal sample loading. Bands were quantitated by ImageJ software for 5 ZAP KO and 5 control cell lines. For each pair of cell lines (KO and control), endogenous ZCCHC3 band intensity was calculated as a percentage of summed Ponceau S-stained bands between the two arrows indicated (bottom right) and normalized to empty vector control (histogram, left).