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. 2012 Oct 1;287(47):39564–39577. doi: 10.1074/jbc.M112.346528

FIGURE 6.

FIGURE 6.

GOLPH3 knockdown decreases PSGL-1-associated C2-O-sLex. A, KG1a cells were transfected with either mock (non-targeting) siRNA, GOLPH3 siRNAs, or C2GnT1 siRNA and cultured for 48–72 h. Cell surface C2-O-sLex and PSGL-1 were analyzed by flow cytometry using mouse anti-C2-O-sLex (CHO-131) IgM and F(ab)2 fragment of goat anti-mouse IgM fluorescein isothiocyanate-conjugated secondary Abs, and rabbit anti-PSGL-1 Abs and F(ab)2 fragment of goat anti-rabbit phycoerythrin-conjugated secondary Abs. GOLPH3 or C2GnT1 siRNA treatment reduced PSGL-1-associated C2-O-sLex from 40.6 to 10.3 and 7.3%, respectively. B, GOLPH3 or C2GnT1 knockdown did not affect the PSGL-1 protein level. C and D, real-time quantitative PCR analysis of C2GnT1 and GOLPH3 in KG1a cells transfected with C2GnT1 or GOLPH3 siRNA, respectively. Knockdown of the mRNA of either gene did not affect the expression level of the other.