Fig. 4

Knocking out (KO) ST8SIA2 eliminates cell surface dPSA and PSA from CHP-134 cells. Cell surface dPSA (A) and polySia (B) on live wild-type (dotted blue line) and the indicated mutants (solid orange lines) detected by FACS using anti-dPSA mAb SEAM 3 and anti-polySia mAb SEAM 12 as primary antibodies. Controls included no primary antibody (gray-filled histograms) and irrelevant IgG2b or IgG2a mAbs, respectively (solid black lines). Fluorescence micrographs (C) of wild-type CHP-134 transfected with an empty plasmid compared to the CRISPR knockout cell lines of the indicated genes. Red fluorescence is anti-dPSA (SEAM 3); blue fluorescence is DAPI DNA staining. Scale bars, 10 μm