Detection of endogenous PTPN22.6 protein in human T cells.

<p><b>A.</b> Protein extract was prepared from HT-29 or Jurkat cells, which were mock (M) transfected or transfected with PTPN22 specific siRNA or scrambled (Sc) siRNA prior to harvest. The extract was analyzed with Western blotting using Lyp antibody. The arrowheads indicate protein species that are not present in HT-29 cells and are suppressed by PTPN22 siRNA. <b>B.</b> 293T cells were transfected with the plasmid vector expressing FLAG-PTPN22.1 or FLAG-PTPN22.6. The transfected cells were then analyzed with Western blotting using PTPN22.6 and Hsp90 antibody. <b>C.</b> Protein extract prepared from Jurkat cells described in <b>A</b> was subjected to immunoprecipitation with anti-Lyp antibody or control IgG. The immunoprecipitate along with protein extract prepared from 293T cells expressing FLAG-PTPN22.6 were analyzed with Western blotting using PTPN22.6 antibody. A fraction of Jurkat cell extract was set aside prior to immunoprecipitation and was probed with Hsp90 antibody to demonstrate equal input. IP and IB stand for immunoprecipitation and immunoblot, respectively. <b>D.</b> Primary human Th cells were harvested from healthy donors and expanded in vitro with anti-CD3/atni-CD28 for 5 days. The cells were then transfected with siRNA and subjected to immunoprecipitation as Jurkat cells described in <b>A</b> and <b>C</b>. The data shown in <b>A</b>, <b>B</b>, and <b>C</b> are representative of at least two independent experiments.