Characterization of ZEBRA protein expression in Jurkat cell lines.

(A) A novel 43-kd protein (denoted ZEBRA) was detected by monoclonal antibody OT20A only in cells transfected with pSV2-neo-WZhet expression plasmid but not control cells transfected with plasmid pSV2-neo. Whole-cell lysates were generated in hypertonic RIPA buffer from COS cells and 2 independently derived Jurkat cell lines transfected with pSV2-neo-WZhet (Z+) and a Jurkat cell line expressing control plasmid (Z−). (B) A novel 43-kd protein was detected in proteins precipitated from Z+ cells but not Z− cells with polyclonal human antiserum (Figure 1A) and probed with OT20A. (C) Z+ and Z− cells were analyzed by immunofluorescence with rabbit polyclonal serum raised against purified ZEBRA protein. Staining was evident primarily in the cytoplasm of Z+ but not Z− cells. Z− and Z+ cells were analyzed by immunofluorescence with a rabbit polyclonal serum raised against purified ZEBRA protein. Staining was evident in the cytoplasm of Z+ but not Z− cells. (D) A novel 43-kd protein was detected with monoclonal antibody OT20A in proteins extracted from Z+ cells but not Z− cells in hypotonic RIPA buffer. Hypotonic lysis buffer was used to extract cytoplasmic proteins selectively from Z+ and Z− cells. Transient transfection of plasmid pC53-SCX3 encoding DNA binding mutant p53 denoted p53N, plasmid pC53-SN3 encoding wild-type p53 denoted p53W, and pCEP4-353 encoding carboxyl terminus deleted p53 denoted p53C did not alter expression of putative 43-kd ZEBRA protein. (E) Z+ Jurkat cells demonstrated increased adherence to plastic ware and an ameboid morphology in comparison to Z− cells grown under identical conditions.