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. 2017 Aug 29;6:e23649. doi: 10.7554/eLife.23649

Figure 2. GDE3 is a GPI-specific PLC that cleaves uPAR in cis.

Figure 2.

(A) (Left) Scheme showing uPAR cleavage in cis or trans. (Right) GDE3-expressing HEK-uPAR cells were mixed with a GDE3-deficient cell population, as indicated. Immunoblot analysis of uPAR in medium and cell lysates indicates that GDE3 acts in cis; mock refers to empty vector-transfected cells. (B) GDE3 expression leads to increased GPI-free suPAR. Conditioned medium from HEK-uPAR cells expressing GDE2 or GDE3 was subjected to Triton X-114 phase separation. suPAR in the aqueous (A) and detergent (D) fractions was analyzed by immunoblotting. (C) GPI-anchor with phospholipase cleavage sites indicated; HONO, nitrous acid. (D) Representative LC-MS ion chromatograms (m/z 259.02–259.03); inositol 1-phosphate peaks in red. HONO-treated suPAR contains inositol 1-phosphate (n = 3, mean ±SEM; *p<0.05).