Posttranslational modification of selectin ligands. (A) Flow cytometric surface staining of CD43a (top), CD43c (middle), and PSGL-1 (bottom) on WT and T-bet^–/– T cells activated under Th1- or Th2-polarizing conditions (gated on live CD4⁺ cells; black = isotype, red = WT, and green = T-bet^–/–). (B) Real-time PCR analysis of mRNA levels of FucTVII expressed in CD4⁺ T cells under Th1- and Th2-polarizing conditions (normalized to β-actin). (C) Real-time PCR analysis of mRNA levels of TPST-1 and TPST-2 expressed in CD4⁺ T cells under Th1-polarizing conditions (normalized to β-actin). □ indicates WT; ▪, T-bet^–/–. (D) Autoradiograph of ³⁵S incorporation into PSGL-1, with or without removal of O- and N-linked glycans. (E) Western blot for PSGL-1 in WT and T-bet^–/– (knock out [KO]) CD4⁺ T cells activated under Th1-polarizing conditions (top panel = PSGL-1 dimer; bottom panel = PSGL-1 monomer). (F) Quantification of protein expression of PSGL-1 either with or without removal of O- and N-linked glycans. Results are expressed as mean ± SEM. WB indicates Western blot.