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. Author manuscript; available in PMC: 2016 Oct 27.
Published in final edited form as: Biochemistry. 2015 Oct 9;54(42):6462–6474. doi: 10.1021/acs.biochem.5b00532

Figure 3.

Figure 3

Recombinant, full-length hPPIP5K1 expresses 1-phosphatase activity. Panel A, Western blot of hPPIP5K1 expressed in Drosophila S3 cells and pulled down with avidin-beads (see Materials and Methods). Panel B, CarboPac HPLC analysis of zero-time incubations of 1 μM 1,5-[3H]InsP8 with 0.69 μg/mL recombinant hPPIP5K1 pulled down from S3 cells with avidin beads (filled symbols); open symbols show the elution of standards of 1-[3H]InsP7 and 5-[3H]InsP7 obtained in parallel runs. Panel C, CarboPac HPLC analysis of assays as described for panel B, except the incubation time was 30 min. Panel D, CarboPac HPLC analysis of assays as described for panel B, except the incubation time was 300 min. Panel E, HPLC analysis of assays as described for panel B, except the incubation time was 30 min, and “control” (no enzyme) avidin beads were added (see Materials and Methods). A Partisphere SAX column was used for these assays, and hence the fraction numbers are not comparable with those in panels B, C, and D.