ZAP colocalizes with L1 ORF1 protein in cytoplasmic granules and interacts with its RNP in an RNA-dependent manner.

<p>(A) EGFP-tagged ORF1p expressed from the construct ORF1-GFP L1-RP colocalizes with V5- tagged ZAP-S in the cytoplasm and SGs of 293T cells. DyLight 650-conjugated secondary antibody was used to detect endogenous eIF3η, a marker of SGs. (B) GFP-tagged ZAP-S (construct GFP-PARP13.2) colocalizes with endogenous ORF1p in cytoplasmic granules of 2102Ep cells. Cell nuclei were stained with Hoechst 33342. (C) EGFP-tagged ORF1p colocalizes with endogenous ZAP in the cytoplasm of 293T cells. (D) L1 RNA tagged with MS2-repeats and expressed from construct 99-PUR-L1-RP MS2-6X colocalizes with GFP-tagged ZAP-S when detected by antisense RNA-FISH probe Cy3-MS2. (E) Exogenously expressed HA-ZAP-L (top panels) and endogenous ZAP (bottom panels) associate with pc-L1-1FH immunoprecipitates from 293T cells. The associations are lost with RNase treatment. (F) FLAG-tagged ZAP-L (ZAP-L-FL) co-IPs untagged ORF1p expressed from construct pc-L1-RP in 293T cells. (G) Human HA-ZAP 1–256, comprising the N-terminal zinc-finger domain only, fails to co-IP with pc-L1-1FH-encoded ORF1p.