Fig. 6. PTPN23 directly interacts with NAP1 via the Bro1 domain.

a NOMO-1 cell lines that constitutively express ALIX-FLAG-BirA*, PTPN23tr-FLAG-BirA*, or PTPN23fl-FLAG-BirA* used in BioID-MS experiment. b Venn diagram showing the intersection and difference of PTPN23 and ALIX interactors. Three independent experiments were performed and included only proteins detected in all three repeats. The targeted binding group was the 65-membered PTPN23tr⁺/PTPN23fl⁺/ALIX⁻ group. c Biotinylation of various BirA* tagged recombinant proteins in NOMO-1 cells. NOMO-1 cell lines stably expressing indicated constructs were treated with 50 μM Biotin for 24 h, followed by streptavidin pulldown. Asterisks mark the self-labeling of BirA* tagged recombinant proteins. d Immunoprecipitation assays of endogenous NAP1 in NOMO-1 stable cell lines expressing indicated constructs. Asterisks mark the Flag-tagged recombinant proteins. e Co-immunoprecipitation of NAP1 and indicated PTPN23 domains or ALIX using proteins purified from E. coli. Each Immunoblots was reproduced three times with similar results for (c–e). f Immunostaining of HA-NAP1-overexpressing HeLa cells with PTPN23 (green) and HA (red) antibodies. Nuclei were counterstained with DAPI (blue). %volume of NAP1 above threshold colocalized 45.7 ± 12.4 (mean ± s.d.). Data were derived from 43 total cells captured in z-stacks across 2 independent transfection experiments; scale bar: 5 μm. g Schematic representation of NAP1 isoforms and constructs. h Co-immunoprecipitation assays of NAP1 constructs and truncated PTPN23 or PTPN23/ALIX chimeras. The plasmids expressing indicated proteins were transfected into HEK 293 T cells. Cells were harvested 48 h after transfection. (n = 3 independent experiments). i Co-immunoprecipitation assays of NAP1 isoforms constructs and TNFR1 in HEK 293 T cells. Cells were harvested 36 h after transfection. (n = 3 independent experiments).