Figure 3: VCP-KMT-mediated trimethylation of VCP in vivo.
From: Lysine methylation of VCP by a member of a novel human protein methyltransferase family
(a) MS-analysis of VCP isolated from mouse tissues. Chromatograms for un-, di- and trimethylated Lys315 in Arg-C-generated peptide VCP(314–322) are shown. Selective ion settings were m/z=542.7727, 556.7884 and 563.7962±6 p.p.m., z=+2. For each tissue, the intensity is normalized to the Kme3 signal. Expected elution time of the Kme0 peptide (arrows) and irrelevant peaks (*, M+1 isotope peak of an unrelated peptide) are indicated. Monomethylated peptide was not detected. (b) Zinc-finger nuclease-mediated disruption of the VCP-KMT gene in human cell lines. In a schematic representation of the human VCP-KMT gene, boxes indicate the coding (grey) and non-coding (light grey) parts of exons, whereas solid black lines indicate introns (not to scale). A blow-up of the sequence at the ZFN target site is shown. ZFN cleavage is expected to occur between the two ZFN-binding sites (uppercase). The genotypes of selected clones harbouring frame shift mutations are shown. U87-MG cells contain only a single copy of the VCP-KMT gene. (c) MS-analysis of VCP isolated from wild-type and VCP-KMT-deficient cell lines. Settings as in (a). For each cell line, the chromatograms are normalized to the highest intensity measured. Kme2 peptides (arrows) and irrelevant peaks (*, M+1 isotope peak of an unrelated peptide) are indicated. (d) Assessment of VCP methylation by western blot. Upper panel, western blot of immunoprecipitated VCP from wild-type and VCP-KMT-deficient cell lines using pan-Kme3 antibody. Lower panel, similar blot of in vitro methylated VCPΔD2 (positive control). A Ponceau S stain of the membrane is shown as loading control.
