HLA class I molecules are physically linked to CD99 molecules in Jurkat cells. (A) Schematic diagram of GST-CD99 fusion proteins prepared for this study. LF indicates long form; SF, short form; IC, CD99 intracytoplasmic region; EC, CD99 extracellular region; TM, CD99 transmembrane region. For GST-val ₃* val 134,135,136 were mutated in Leu, Leu, Ile, and for GST-val ₅* mutations of val 131 and 138 to Leu were added. (B) The short form of CD99 can bind HLA class I molecules. GST pull-down assays were performed on Jurkat cell lysates using the indicated GST-CD99 fusion proteins (above lanes 1-6 and 7-14). After extensive washings, bound proteins were separated by SDS-PAGE and immunoblotted (IB) with the “H300” Ab directed against HLA class I molecules. The whole-cell lysates used for HLA class I binding analysis are shown on the immunoblots in lanes 2 and 14. (C) Val 134-136 residues of the transmembrane and HLA class I binding domains. GST pull-down assays were performed using GST-CD99 fusion proteins extending into the transmembrane region GST-Val ₅ + IC). Mutations to Leu or Ile were introduced at Val 131, 134-136, and 138 (GST-Val _3* + IC and GST-Val _5* + IC). (D) Expression of HLA class I by transfectants showing CD99 long form mutated on the valines 134-136 (CD99 LF Val) compared with CD99 long form WT (CD99LF_wt) transfectants and CD99 negative cells (CD99_neg).