Figure 6
Figure 6. The transmembrane region of CD99 associates with the p230 GRIP domain region. (A) Colocalization of HLA class I molecules and p230/golgin-245. Jurkat T cells were fixed, permeabilized, and stained for IFM. Trafficking markers were visualized by indirect fluorescence using specific mAbs, detected with a Texas Red–conjugated horse anti–mouse IgG. HLA class I molecules were detected with the FITC-“W6/32” mAb. Bip/GRP78: a major chaperone of the ER lumen; LAMP1: a glycoprotein of lysosome membranes; GM130: a Golgi matrix protein, also associated with the cis-Golgi; GS27/membrin: a Golgi-associated SNARE which acts in medial- to trans-Golgi protein movement; Syntaxin 11: a t-SNARE from the post-Golgi; Rab8: involved in the regulation of vesicular transport from the TGN to the plasma membrane; p230/golgin-245: a protein of the TGN structure. (B) Immuno-electron microscopy identifies colocalization of p230 and CD99. Consecutive immuno-gold double labeling of Jurkat cells: detection of p230 (anti-p230 mAb) and protein A coupled to 15-nm gold particles followed by labeling with CD99 (O662 mAb) and protein A coupled to 10-nm gold particles. The arrows point to p230 and CD99 colocalization areas. (C) GST-CD99 fusion proteins were incubated with Jurkat cell extracts. Bound proteins were detected with a p230-specific antibody. The whole-cell lysates used for the analysis of p230 binding are shown in lanes 4 and 9 of the immunoblot. LAMP1 staining was also used as a control. (D) GST-p230 fusion proteins expressing only the proline-rich domain (1-270aa) or the C-terminal region encompassing the GRIP domain of p230 (2000-2230aa) were incubated with Jurkat cell extracts. The eluted material was analyzed by Western blotting for CD99 using the “12E7” mAb. The whole-cell lysate is shown in lane 4. GM130 staining was also used as a control. (E) Jurkat cells were lysed in 0.5%TTX lysis buffer. Samples were immunoprecipitated with the “O662” CD99 mAb. Immunoprecipitates were analyzed by Western blot against HLA class I (H300 mAb) and anti-p230.

The transmembrane region of CD99 associates with the p230 GRIP domain region. (A) Colocalization of HLA class I molecules and p230/golgin-245. Jurkat T cells were fixed, permeabilized, and stained for IFM. Trafficking markers were visualized by indirect fluorescence using specific mAbs, detected with a Texas Red–conjugated horse anti–mouse IgG. HLA class I molecules were detected with the FITC-“W6/32” mAb. Bip/GRP78: a major chaperone of the ER lumen; LAMP1: a glycoprotein of lysosome membranes; GM130: a Golgi matrix protein, also associated with the cis-Golgi; GS27/membrin: a Golgi-associated SNARE which acts in medial- to trans-Golgi protein movement; Syntaxin 11: a t-SNARE from the post-Golgi; Rab8: involved in the regulation of vesicular transport from the TGN to the plasma membrane; p230/golgin-245: a protein of the TGN structure. (B) Immuno-electron microscopy identifies colocalization of p230 and CD99. Consecutive immuno-gold double labeling of Jurkat cells: detection of p230 (anti-p230 mAb) and protein A coupled to 15-nm gold particles followed by labeling with CD99 (O662 mAb) and protein A coupled to 10-nm gold particles. The arrows point to p230 and CD99 colocalization areas. (C) GST-CD99 fusion proteins were incubated with Jurkat cell extracts. Bound proteins were detected with a p230-specific antibody. The whole-cell lysates used for the analysis of p230 binding are shown in lanes 4 and 9 of the immunoblot. LAMP1 staining was also used as a control. (D) GST-p230 fusion proteins expressing only the proline-rich domain (1-270aa) or the C-terminal region encompassing the GRIP domain of p230 (2000-2230aa) were incubated with Jurkat cell extracts. The eluted material was analyzed by Western blotting for CD99 using the “12E7” mAb. The whole-cell lysate is shown in lane 4. GM130 staining was also used as a control. (E) Jurkat cells were lysed in 0.5%TTX lysis buffer. Samples were immunoprecipitated with the “O662” CD99 mAb. Immunoprecipitates were analyzed by Western blot against HLA class I (H300 mAb) and anti-p230.

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