KPL1 recognizes the tyrosine sulfation motif of PSGL-1. (A) 300.19 cells were transiently transfected with either full length PSGL-1 (top) or a mutated form of PSGL-1 referred to as FFFE/PSGL-1 (bottom) in which the tyrosines at positions 5, 7, and 10 were replaced with phenylalanine. Twenty-four hours posttransfection, the 300.19 cells were stained with either a negative control antibody, KPL1, PL2, or PSL275. PL2 and PSL275 recognize both the full length and mutated form of PSGL-1, whereas KPL1 was able to interact with full length PSGL-1 but did not recognize FFFE/PSGL-1. (B) Arylsulfatase treatment of whole cell lysates abrogates binding of KPL1. Whole cell lysates were prepared as described in Materials and Methods and treated with 1U of arylsulfatase, and Western blotting was performed as described for Fig 1, using KPL1 (left) or PL2 (right).