FIGURE 2.
The expression of B7-2C molecules by cultured non-neoplastic SGEC was found to associate with low surface expression of the full-length B7-2A protein. A, Representative examples of SGEC lines manifesting high (left) or low (right) constitutive surface expression of the B7-2A protein, as assayed by flow cytometry using a PE-conjugated anti-B7-2 mAb (FUN.1) (open histogram). Staining with an isotype-control Ab (negative control) (gray-shaded histogram) is also shown. B, Immunoblotting analysis of surface expression of the B7-2A and B7-2C protein isoforms on representative SGEC lines manifesting high or low constitutive surface B7-2A protein expression (as revealed by flow cytometry). SGEC lines used are the same as shown in A. Surface proteins of cells were biotinylated, and B7-2 proteins were subjected to immunoprecipitation using the anti-cytoplasmic B7-2 Ab C19 (that recognizes the cytoplasmic tail of both B7-2A and B7-2C isoforms). An irrelevant Ab (Ct. Ab) was applied for the evaluation of nonspecifically immunoprecipitated proteins (negative control). Immunoprecipitated proteins were analyzed by SDS-PAGE, and biotinylated molecules were visualized by immunoblotting with alkaline phosphatase-conjugated streptavidin. Among SGEC lines with high and those with low constitutive surface B7-2A expression, significantly higher constitutive expression of B7-2C proteins was detected in SGECs with low B7-2A expression. Compared with B7-2A and B7-2C protein isoforms expressed by CHO-B7-2A/C transfectants, those of SGEC correspond to glycoprotein bands of higher molecular masses, compatible with heavier glycosylation status. The experiments performed are representative of three SGEC lines from each group (high and low B7-2A expression, respectively). CHO-mock/mock (m/m) and CHO-B7-2A/C transfectants were used as negative and positive control cell lines, respectively. An irrelevant protein band that is detected by the alkaline phosphatase-conjugated streptavidin reagent (asterisk), which was applied for the visualization of biotinylated proteins.

The expression of B7-2C molecules by cultured non-neoplastic SGEC was found to associate with low surface expression of the full-length B7-2A protein. A, Representative examples of SGEC lines manifesting high (left) or low (right) constitutive surface expression of the B7-2A protein, as assayed by flow cytometry using a PE-conjugated anti-B7-2 mAb (FUN.1) (open histogram). Staining with an isotype-control Ab (negative control) (gray-shaded histogram) is also shown. B, Immunoblotting analysis of surface expression of the B7-2A and B7-2C protein isoforms on representative SGEC lines manifesting high or low constitutive surface B7-2A protein expression (as revealed by flow cytometry). SGEC lines used are the same as shown in A. Surface proteins of cells were biotinylated, and B7-2 proteins were subjected to immunoprecipitation using the anti-cytoplasmic B7-2 Ab C19 (that recognizes the cytoplasmic tail of both B7-2A and B7-2C isoforms). An irrelevant Ab (Ct. Ab) was applied for the evaluation of nonspecifically immunoprecipitated proteins (negative control). Immunoprecipitated proteins were analyzed by SDS-PAGE, and biotinylated molecules were visualized by immunoblotting with alkaline phosphatase-conjugated streptavidin. Among SGEC lines with high and those with low constitutive surface B7-2A expression, significantly higher constitutive expression of B7-2C proteins was detected in SGECs with low B7-2A expression. Compared with B7-2A and B7-2C protein isoforms expressed by CHO-B7-2A/C transfectants, those of SGEC correspond to glycoprotein bands of higher molecular masses, compatible with heavier glycosylation status. The experiments performed are representative of three SGEC lines from each group (high and low B7-2A expression, respectively). CHO-mock/mock (m/m) and CHO-B7-2A/C transfectants were used as negative and positive control cell lines, respectively. An irrelevant protein band that is detected by the alkaline phosphatase-conjugated streptavidin reagent (asterisk), which was applied for the visualization of biotinylated proteins.

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