![Fig. 2. The activation epitope recognized by MEM-148 is present on a 65- to 70-kd protein. / (A) Immunoprecipitation from surface-biotinylated PBLs. The cells were biotinylated either before or immediately after 2-hour PMA treatment; control cells were biotinylated and left untreated. The cell lysates (1% NP40) were immunoprecipitated by means of mAbs to the indicated molecules (MEM-148; 6.7, CD18; MEM-83, CD11a; MEM-170, CD11b; LeuM5, CD11c; B2M-02, β2-microglobulin) and the immunoprecipitates subjected to SDS-PAGE followed by Western blotting using streptavidin-peroxidase to reveal biotinylated proteins. (B) Isolated neutrophils or monocytes were activated with PMA as in Figure1B or left untreated for 120 minutes (Control) and at the indicated time points (15-120 minutes) analyzed by SDS-PAGE and immunoblotting using MEM-148 for detection (top panels); the bottom panel shows quantitative densitometric evaluation (plotted for the respective lanes as ratio of the signal from the 65- to 70-kd zone [closed arrowhead] versus the upper 78- to 96-kd CD18 zone [open arrowhead]). (C) Western blotting analysis of PBLs after various activating treatments. PBLs were activated using the indicated stimuli for 2 hours (for the different activation protocols see “Materials and methods”). The blot was immunoperoxidase-stained with the standard CD18 mAb MEM-48; a very similar, albeit weaker, staining pattern was obtained also with MEM-148 (not shown). Open and closed arrowheads in all parts of the Figure 2 indicate the zones corresponding to the uncleaved CD18 species (78-96 kd) and the 65- to 70-kd fragment, respectively.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/98/5/10.1182_blood.v98.5.1561/5/h81711466002.jpeg?Expires=1735554060&Signature=LU2SNN15yIwVWQGoFtb68eUN0uHb6~DrPXlEKn70JglLvHmnkkDeBNPjrd8UMnZ42XWTBJa1Mx~PehULdiV8gbqqRjB9Fwcoi6Y1Qrjw2FVzXHlAV3CLzoDrmIsGHYPblyggYmxeiLPxiTjyJ5hLqrndtWaxu5fW9Jy9SJEK1Jgl8jTrQex~1E3gg3Bw0pa9gTiQuJYh4w5dj6r9MduxspwHEjOms5px0Qtq~b8Pa0S5vn1i3WSALGJr552UhOWBUU-5QF3i82IEpMgFZtPMVwqlibwf-IEfBZNmJ1Ay4z4sgJSqK7Y8LBFW0ISnmvHZSq86iR2GFdNvW-l0jhR75A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The activation epitope recognized by MEM-148 is present on a 65- to 70-kd protein.
(A) Immunoprecipitation from surface-biotinylated PBLs. The cells were biotinylated either before or immediately after 2-hour PMA treatment; control cells were biotinylated and left untreated. The cell lysates (1% NP40) were immunoprecipitated by means of mAbs to the indicated molecules (MEM-148; 6.7, CD18; MEM-83, CD11a; MEM-170, CD11b; LeuM5, CD11c; B2M-02, β2-microglobulin) and the immunoprecipitates subjected to SDS-PAGE followed by Western blotting using streptavidin-peroxidase to reveal biotinylated proteins. (B) Isolated neutrophils or monocytes were activated with PMA as in Figure1B or left untreated for 120 minutes (Control) and at the indicated time points (15-120 minutes) analyzed by SDS-PAGE and immunoblotting using MEM-148 for detection (top panels); the bottom panel shows quantitative densitometric evaluation (plotted for the respective lanes as ratio of the signal from the 65- to 70-kd zone [closed arrowhead] versus the upper 78- to 96-kd CD18 zone [open arrowhead]). (C) Western blotting analysis of PBLs after various activating treatments. PBLs were activated using the indicated stimuli for 2 hours (for the different activation protocols see “Materials and methods”). The blot was immunoperoxidase-stained with the standard CD18 mAb MEM-48; a very similar, albeit weaker, staining pattern was obtained also with MEM-148 (not shown). Open and closed arrowheads in all parts of the Figure 2 indicate the zones corresponding to the uncleaved CD18 species (78-96 kd) and the 65- to 70-kd fragment, respectively.
