ADAMTS Sol narae cleaves extracellular Wingless to generate a novel active form that regulates cell proliferation in Drosophila

In all western analyses, blotting antibodies are indicated at the bottom of each panel. Fractions and the source of cells transfected with different constructs are written at the top of panels. S2 cells are derived from a macrophage-like lineage of fly embryonic cells78, and express neither Sona nor Wg10,19,33. Therefore, UAST-GFP-wg and UAST-sona-HA cDNA constructs were expressed by actin-Gal4 driver in S2 cells. HA is the preparation from cells transfected with the control UAST-HA vector. The pound signs (#) indicate the cleaved Wg fragments in the absence of Sona. These fragments may be generated by degradation during sample preparation or by some Wg-specific proteases endogenously expressed in S2 cells. a Full-length and processed forms of Sona-HA in cell extract (CX), SNΔ, and P100 fractions. The P100 fraction was four times more concentrated than the SNΔ fraction, and the equal volumes of concentrated samples were loaded for the blot. Therefore, the actual amount of Sona in the SNΔ fraction should be four times more than the one in the blot. The SNΔ fraction always contained more Sona than the P100 fraction. Full-length Sona and active Sona are marked with red arrows and black arrow, respectively. b Full-length GFP-Wg (GFP-WgFL) in CX, SNΔ, and P100 fractions. SNΔ and P100 fractions were concentrated equally and the same volumes were loaded. GFP-Wg was more abundant in the SNΔ fraction than the P100 fraction (red arrow)19,33. c Verification of CX, P100, SN0, and SNΔ fractions using the ER marker Calnexin and the exosomal markers Alix and Syntaxin 1A (Syx1A). d Diameter and number of vesicles in P100 fractions from S2 sona-HA cells measured by NTA. e A immuno-EM image of Sona-HA in the P100 fraction with anti-HA antibody. The diameter of vesicles with Sona-HA is about 120？nm. f–f” GFP-WgFL (83？kDa) and a GFP-Wg fragment (65？kDa) are indicated with red and black arrows, respectively. g, h The black arrow and the arrowhead indicate 65 and 60？kDa GFP-Wg fragments, respectively. The same blot was used for both (g) and (h). i A blot in (i) was exposed longer in (i’). The 41？kDa and the 23？kDa fragments are marked by the black arrows and the red arrowhead, respectively. Non-tagged WgFL are marked by red arrows. j The 23？kDa Wg-3XHA fragment are marked with the red arrowhead. This fragment was readily detected because it has three times HA epitope than 4D4 epitope. k Domain structures of full-length and cleaved forms of Wg (modified from a report79). Two cleavage sites (L1, L2) are indicated with scissors. The 4D4 epitopes are marked with re