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. 2015 Apr 13;209(1):85–95. doi: 10.1083/jcb.201409064

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© 2015 Nakatsu et al.

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Figure 6. — Sac2 is an inositol 4-phosphatase. (A) GFP, GFP-Sac2, and GFP-Sac2^D460N were transiently overexpressed in Expi293 HEK cells and semi-purified by immunoprecipitation using anti-GFP antibody. The presence of inositol-phosphatase activity in the immunoprecipitated material was assessed with a malachite-based assay. Data shown represent mean ± SD (error bars). (B) Lysates of COS-7 cells expressing GFP, GFP-Sac2^WT, or GFP-Sac2^D460N were subjected to anti-GFP immunoprecipitation, and immunoprecipitates were processed by immunoblotting to assess the enrichment of GFP and GFP fusion proteins (bottom), of inositol phosphatases (OCRL, INPP5B, and synaptojanin 1), and of control proteins (epsin 1 and Erk1/2). Note the enrichment of OCRL and INPP5B in anti-GFP immunoprecipates from COS7 cells expressing GFP-Sac2^WT or GFP-Sac2^D460N. (C) GST-Sac^WT and GST-Sac^D460N were expressed in bacteria, purified on glutathione Sepharose beads, and analyzed for inositol phosphatase activity against the seven PIs via the malachite assay. Data shown represent mean ± SD (error bars).