Cellular and Molecular Life Sciences
The amino terminus extension in the long Dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus
Daniela Justa-Schuch, Ulrike Möller and Ruth Geiss-Friedlander *
Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Goettingen,
Humboldtallee, 37073 Goettingen, Germany
*To whom correspondence should be addressed: Ruth Geiss-Friedlander, Department of Molecular
Biology, Georg-August-University of Goettingen, Humboldtallee 23, 37073 Goettingen, Germany,
Phone: (49) 551 395984 Fax: (49) 551 395960, E-mail: rgeiss@gwdg.de
Supplementary Fig. 1 A Complete protein sequence alignment of DPP9-L and DPP9-S showing that both isoforms only differ in their N-termini. B DPP9 was silenced in HeLa cells using four different siRNAs and a non-targeting control siRNA. 10 µg of the p repared cell extracts were analyzed with the
Abcam anti DPP9 (total) antibodies. Tubulin was used as loading control. These are the same samples as those analyzed in Fig. 1F with the DPP9 (long) antibody. Three major bands are observed in the total cellular extracts with the anti DPP9 (total) antibody: ca. 95, 120 and 200 kDa. Note that two bands

corresponding in size to 95 kDa and 120 are missing in DPP9-silenced cells, suggesting that they are truly DPP9. The nature of the band at 120 kDa is currently unclear and may represent a modification of
DPP9. Importantly, the band running at 200 kDa does not disappear upon DPP9-silencing in the corresponding cell extract, and thus we conclude that it is not DPP9
Supplementary Fig. 2 A Representative confocal images of negative control experiments for indirect immunofluorescences, in which no primary antibody was used during the procedure to estimate background staining of various secondary antibodies used in this study (anti rabbit Alexa Fluor594, anti goat Alexa Fluor488, anti mouse Alexa Fluor488). These controls were included in each immunofluorescence experiment performed. Hoechst staining was applied to visualize the nucleus. B
Activities of nuclear extracts from overexpressing cells shown in figure 3D. 10 μg of nuclear extracts from DPP9-L or DPP9-S overexpressing HeLa cells were analyzed for hydrolysis of GP-AMC. Shown is a representative Michaelis-Menten analysis performed in triplicates, including error bars. The experiment was repeated at least three times C.
Localization of DPP9-S and DPP9-L is not altered upon inhibition of CRM1-dependent nuclear export. Chart showing the subcellular distribution of
DPP9-L and DPP9-S in HeLa cells after leptomycin B treatment. HA-tagged DPP9-L or DPP9-S was overexpressed in HeLa cells for 48 h. Prior to fixation, cells were treated for 4 hours with leptomycin B
(5 nM) or for control with EtOH, and subsequently subjected to indirect immunofluorescence detecting the HA tag. Transfected cells were grouped into N > C (stronger signals in the nucleus) and C > N
(stronger signals in the cytosol)

Supplementary Fig. 3 Full scans of Western blots shown in Fig. 5B A Complete scan of the subcellular fractionation experiment shown in Fig. 5B, left panel. Note that protease inhibitors were omitted from the fractionation procedure to enable subsequent DPP9 activity measurements in the various fractions. Asterisk labels putative DPP9 degradation product. B Full scan of the fractionation experiment shown in Fig. 5B, right panel. Note that no protease inhibitors were used during the fractionation procedure to enable subsequent DPP9 activity measurements in the various fractions. * labels putative DPP9 degradation product, ** labels putative modification product of DPP9

Supplementary Fig. 4 A Complete Western Blot membrane of a representative fractionation experiment decorated with antibodies against VDAC that is shown in Fig. 6 including purified mitochondria from HEK293T cells as a VDAC positive control (PC). B Complete Western blot of the membrane decorated with antibodies against DPP8 that is shown in Fig.6. C DPP8 or DPP9 were silenced in HeLa cells by using specific siRNAs and a non-targeting control siRNA. Total cell extracts were prepared and tested for DPP8 expression (10 µg extract per lane) . Note that while the corresponding DPP8 band at a size of approximately 95 kDa is missing in DPP8-silenced cells, bands of higher molecular weights do not disappear upon DPP8-silencing in the corresponding cell extract.
Tubulin was used as loading control D The graph shows Michaelis-Menten assays performed as triplicates of 12.5 nM recombinant DPP9-L incubated in the different fractionation buffers (Hypotonic buffer, Membrane buffer and Total buffer) and as a control in the usual reaction buffer TB + 0.02%
Tween illustrating the effect of the detergents in the buffers on DPP activity. E Elution profiles of 200 nM recombinant DPP9-L incubated in TB buffer or Total buffer, respectively, on a gel exclusion chromatography, using analytical Superdex S200. As size standard Bio-Rad´s gel filtration standard was used. Profiles were generated using Prism software with ml on x-axis and UV absorbance (mAU
280 nm
) on y-axis. The arrow points to a second peak corresponding to elution of high molecular weight proteins. This fraction may contain aggregated or unfolded DPP9-L in the Total buffer

Supplementary Fig. 5 Defined concentrations of recombinant DPP9-S or DPP9-L and defined concentrations of cellular extracts were analyzed on SDS PAGE with the Abcam DPP9 antibody.