Fig. 3
OUM1 mainly locates in cytoplasm and directly binds to PTPRZ1 protein to enhance PTP activity. a, b OUM1 mainly located in the cytoplasm in OCM1a and OM431 cells. U2 RNA: positive control for nuclear RNA. c Schematic of the regions responsible for binding between OUM1 and PTPRZ1. Sites 2 and 6: different detection locations for RNA ChIP. d, e The interaction of OUM1 and PTPRZ1 was detected. After OUM1 knockdown, the binding between PTPRZ1 and OUM1 was abolished at the primer 2 region and decreased at the primer 6 region in OCM1a-shOUM1 cells. Input: total RNA reverse transcribed and amplified without incubation with the PTPRZ1 antibody. IgG: negative control. f Gray level analysis conducted to quantify the enrichment of OUM1 on PTPRZ1. g, h PTP activity was detected in OCM1a and OM431 cells. The amount of phosphate indicates phosphatase activity. The amount of phosphate decreased from 1.4 pmol PO4/min/μg and 1.5 pmol PO4/min/μg to 1.0 pmol PO4/min/μg and 0.9 pmol PO4/min/μg when P1 and P2 were added, respectively, after OUM1 knockdown in OCM1a cells. In OM431 cells, these values decreased from 3.2 pmol PO4/min/μg and 3.7 pmol PO4/min/μg to 2.1 pmol PO4/min/μg and 2.0 pmol PO4/min/μg when P1 and P2 were added, respectively, after OUM1 knockdown. i Phosphate content increased significantly after synthesized OUM1 RNA was added to the PTP reaction system at increasing concentrations of 1 μg, 5 μg and 10 μg; at these concentrations, the phosphate content was approximately 2.6 pmol PO4/min/μg, 4.2 pmol PO4/min/μg, and 5.2 pmol PO4/min/μg, respectively, in OCM1a cells and 1.8 pmol PO4/min/μg, 2.9 pmol PO4/min/μg, and 4.0 pmol PO4/min/μg, respectively, in shOUM1-OCM1a cells. E Extract, protein extracted from OCM1a, OM431, shOUM1-OCM1a or shOUM1-OM431 cells. P1 and P2 indicate two chemically synthesized phosphopeptides, Tyr phosphopeptides 1 (END(pY)INASL) and 2 (DADE(pY)LIPQQG). SOV (V): Na3VO4, a PTP inhibitor that inhibits PTP activity and is used as positive control. *P < 0.05