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Fig. 2. MK specifically interacts with MK-TRAP in vitro. (A) MK-TRAP expression in COS7 cells transiently overexpressing MK-TRAP or in the G401-derived M1 cell line stably overexpressing MK-TRAP. The arrowhead indicates the secreted form of MK-TRAP; the arrow indicates the pre-secreted form of MK-TRAP; C, cell lysate; M, medium. (B) MK-TRAP-containing medium was mixed with MK-containing medium or control medium, and MK was pulled down with anti-MK antibody. MK-TRAP was detected with anti-HA antibody. (C) MK was pulled down from cellular extracts or medium of the M1 cell line. MK-TRAP was detected in the immunoprecipitates. (D) Expression of MK and MK tagged with a Myc epitope (designated as MK-myc) in COS7 cells transiently transfected with each construct. (E) Cellular extracts or medium from COS7 cells co-transfected with both MK-myc and MK-TRAP were immunoprecipitated with an anti-HA antibody and subsequently detected with anti-Myc antibody, and vice versa. The arrowhead indicates the secreted form of MK-TRAP; the arrow indicates the pre-secreted form of MK-TRAP. (F) MK-TRAP-containing medium was mixed with human recombinant PTN (left panel), bFGF (middle panel) or PDGF-BB (right panel). PTN, bFGF or PDGF-BB was immunoprecipitated with anti-PTN, anti-bFGF or anti-PDGF-BB, respectively. MK-TRAP in each precipitate was detected with anti-HA antibody. (G) Exogenous proteins (10 µg of MK, PTN, bFGF or PDGF-BB) were added to a medium containing MK-TRAP and MK-myc to detect their effects on the binding between MK-TRAP and MK-myc. MK-TRAP was pulled down with anti-HA antibody, and MK-myc in the precipitates was detected using anti-Myc antibody. (H) sLRP3-containing medium was mixed with MK. MK was then pulled down with anti-MK antibody, and sLRP3 in the precipitates was detected with anti-HA antibody.
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