Construct pc-L1-1FH successfully immunoprecipitates basal L1 RNP complexes (ORF1p, ORF2p, and L1 RNA) from 293T cell lysates following α-FLAG purification.

<p>Detection in purified immunoprecipitates of (A) FLAG-HA-tagged ORF1 protein, (B) ORF2 protein, (C) L1 RNA detected by RT-PCR, and (D) ORF2 reverse transcriptase activity, assayed as described by Kulpa et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002941#pgen.1002941-Kulpa1" target="_blank">[35]</a>. (E–I) Endogenous and ectopically expressed MOV10 protein and L1 ORF1p associate in multiple cell lines. (E) Immunoprecipitation of V5-tagged MOV10 by FLAG-tagged pc-L1-1FH depends upon the presence of RNA (lanes 2 and 3). A double point mutation in ORF1 of pc-L1-1FH known to inhibit RNA-binding, prevents efficient co-IP of MOV10 protein (lane 4). Removing the FLAG-HA-tag from pc-L1-FH (pc-L1-RP) prevents IP of the L1 RNP and MOV10 protein on α-FLAG agarose (lane 5). (F) pc-L1-1FH-generated RNPs associate with endogenous MOV10 protein in 293T cells. (G) Transfected V5-tagged MOV10 and cold shock domain protein YBX1, but not fibrillarin (FBL) or empty vector, immunoprecipitate endogenous ORF1p from 2102Ep cells. (H) α-ORF1 (AH40.1) antibody co-IPs endogenous MOV10 protein from 2102Ep cells (lane 1). Lane 3: lysate of 293T cells transfected with MOV10-V5-His₆ WT as a marker for MOV10 protein. (I) Similarly, immunoprecipitation using α-MOV10 antibody yields endogenous ORF1p (lane 1). Lane 4: α-FLAG-tag IP from 293T cells transfected with pc-L1-1FH as a marker for ORF1p.