Overexpression of ADAM17 in KO cells, the effect of flag insertion and biophysical characterization of purified proteins.

(A) Over-expression of pig, mouse, human ADAM17 (+pADAM17, +mADAM17, +hADAM17) and hADAM17/H405D mutant in ADAM17-KO cells was detected using a polyclonal antibody against hADAM17 by immunofluorescence. Nuclei were stained by DAPI. Size bars indicate 100 μm. (B) Indicated cells were infected with CSFVpp-SXCDK and the titer was measured as FFU/ml. The lower limit of detection was 10 FFU/ml (dashed line). Asterisks indicate samples below the limit. (C) Overexpression of flag-tagged pig, chicken and zebrafish ADAM17 (+pADAM17-F, +cADAM17 and +zfADAM17) was detected by western blot analysis using anti-Flag antibody. (D) Biophysical characterization of sE2 and sMP proteins. Gel filtration profiles of sE2 (left) and sMP (right) proteins were analyzed by size-exclusion chromatography on a Superdex 200 10/300 GL column. The absorbance curves at 280 nm and the SDS-PAGE separation profiles of the pooled samples are shown.

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