Fig. 6

Verification of processing of human proteins by HERV-K(HML-2) Protease in vivo. Human candidate proteins and HML-2 Pro were co-expressed in HeLa cells in vivo and detected by Western blot using antibodies as indicated. For each blot, the leftmost lane is a control co-transfected with a plasmid encoding a candidate protein and either a GFP-encoding plasmid or empty phCMV, pcDNA6 myc/his B, or pcDNA5 FRT/TO vector, depending on GFP-Pro or (sole) Pro co-expressed in the experiment (see below). Candidate protein co-expressed with wild-type Pro (pro-wt) and mutant Pro (pro-mut) were loaded in lanes 2 and 3 each. Pro was expessed as either (sole) Pro or EGFP-Pro. Blots were probed with α-HA, α-GFP, α-Pro, or an α-HSP90 antibody as indicated. Full-length candidate protein and processing products are indicated by arrows and arrowheads, respectively (see below). A Representative results from control experiments co-expressing HSP90AA1 with either HML-2 Pro or EGFP-Pro. Relevant blot regions are shown. When expressing pro-wt and pro-mut, HML-2 Pro can be detected as approximately 18 kDa and 19 kDa protein bands representing self-processed and unprocessed products, respectively, Pro (a, bottom blot). When HML-2 Pro is expressed as EGFP-Pro-wt or EGFP-Pro-mut fusion protein, proteins of approximately 30 kDa and 47 kDa, representing processed and unprocessed EGFP(−Pro) can be detected with an α-GFP antibody (b, middle blot). Unprocessed EGFP-Pro(−mut) and self-processed Pro of approximately 50 kDa and 18 kDa, respectively, can be detected when using an α-Pro antibody (b, bottom blot; c). B. Selected Western blot results from co-expression of candidate proteins and HML-2 Pro. Candidate proteins were tagged with N- or C-terminal epitopes and detected with respective epitope-specific antibodies as indicated. Note the more or less complete reduction of amounts of full-length candidate protein (arrows), and sometimes processing products (arrow heads), in lanes with co-expressed HML-2 Pro. Note in panel Aa and Ab that the same processing product was detected for HSP90AA1 in vitro and in vivo (the HSP90AA1 in vitro result is shown again in Ad for the sake of convenience). Also compare in vitro and in vivo results for C15orf57 and MAP2K2 as additional examples of similar sized processing products. Molecular masses of co-migrating marker proteins are indicated. Note that the α-Pro Western blot result shown for CIAPIN1-HA is extracted from the Western blot shown in Ac. See Additional file 2: Figure S3 for loading controls as well as more examples of proteins processed by HML-2 Pro in vivo. C. Graphical depictions of candidate proteins and predictions of processing products as observed when co-expressing candidate proteins and HML-2 Pro-wt in vivo. Numbers of amino acids and corresponding molecular mass (kDa) are indicated by scales at the top and by the length of lines for each protein. Positions of cleavage sites, as identified by TAILS experiments at pH 5.5 and pH 7, are indicated by triangles for each protein. Dashed lines indicate molecular masses of processing products and take into account whether the candidate protein was expressed with an N-terminal or a C-terminal epitope tag. Note the overlap between predictions and molecular masses of processing products observed in vivo