Figure 2.
Figure 2. Bicarbonate transport activity and cell surface expression of wild-type, S725R, or S725A mutants in Band 3 or membrane-domain constructs. HEK293 cells were grown on glass coverslips and transiently transfected with complementary DNA plasmids encoding the full length or membrane-domain constructs indicated. (A) Cells were loaded with the pH-sensitive dye, BCECF-AM, and fluorescence was monitored as cells were alternately perfused with chloride-containing Ringer's buffer (black bars) or chloride-free Ringer's buffer (white bars). HCO3− transport rates were monitored by the initial rate of change of intracellular pH (pHi) induced upon switching to chloride-free medium. Perfusion solutions were bubbled with 5% CO2. (B) (Top) HCO3− transport activity of WT and mutant Band 3 or membrane-domain constructs were corrected for background activity of vector-transfected cells, normalized to protein expression level, and expressed as percentage of wild-type rate. Error bars indicate standard errors of the mean (n = 3). *Significant difference in transport rate from WT-Band 3 or membrane-domain construct. (Bottom) Cell lysates from cells used for transport experiments were probed for Band 3 with IVF12 monoclonal antibody and for endogenous transcription factor IID (TFIID) on immunoblots. (C) Immunofluorescence localization of HA-tagged Band 3 membrane domain and S725R and S725A mutants in transfected HEK cells. We transfected 2 µg of mdAE1 WT and mutants by lipofectamine LTX & PLUS for 24 hours. Fixed HEK cells were incubated with subsaturating amounts of mouse anti-HA (1/500 dilution) antibody, followed by anti-mouse immunoglobulin G (IgG) Alexa 488 (green) to detect cell surface AE1. Then cells were washed, permeabilized by Triton X-100, and incubated with a second aliquot of mouse anti-HA antibody (1/500 dilution), followed by anti-mouse IgG-Cy3 (red) antibody to detect total AE1 expression. Cell images were examined using an LSM 510 confocal microscope. Colocalization (yellow) was calculated by Image J JACoP program using Pearson’s and Manders’s coefficients. Column A indicates cell surface mdAE1 (HA-Alexa 488 in green); column B indicates total mdAE1 (HA-cy3 in red); and column C indicates colocalization of cell surface mdAE1 and total mdAE1 (in yellow) on the cell surface. The average Pearson’s coefficients are WT 0.741, S725A 0.661, and S725R 0.419. TFIID, transcription factor IID.

Bicarbonate transport activity and cell surface expression of wild-type, S725R, or S725A mutants in Band 3 or membrane-domain constructs. HEK293 cells were grown on glass coverslips and transiently transfected with complementary DNA plasmids encoding the full length or membrane-domain constructs indicated. (A) Cells were loaded with the pH-sensitive dye, BCECF-AM, and fluorescence was monitored as cells were alternately perfused with chloride-containing Ringer's buffer (black bars) or chloride-free Ringer's buffer (white bars). HCO3− transport rates were monitored by the initial rate of change of intracellular pH (pHi) induced upon switching to chloride-free medium. Perfusion solutions were bubbled with 5% CO2. (B) (Top) HCO3− transport activity of WT and mutant Band 3 or membrane-domain constructs were corrected for background activity of vector-transfected cells, normalized to protein expression level, and expressed as percentage of wild-type rate. Error bars indicate standard errors of the mean (n = 3). *Significant difference in transport rate from WT-Band 3 or membrane-domain construct. (Bottom) Cell lysates from cells used for transport experiments were probed for Band 3 with IVF12 monoclonal antibody and for endogenous transcription factor IID (TFIID) on immunoblots. (C) Immunofluorescence localization of HA-tagged Band 3 membrane domain and S725R and S725A mutants in transfected HEK cells. We transfected 2 µg of mdAE1 WT and mutants by lipofectamine LTX & PLUS for 24 hours. Fixed HEK cells were incubated with subsaturating amounts of mouse anti-HA (1/500 dilution) antibody, followed by anti-mouse immunoglobulin G (IgG) Alexa 488 (green) to detect cell surface AE1. Then cells were washed, permeabilized by Triton X-100, and incubated with a second aliquot of mouse anti-HA antibody (1/500 dilution), followed by anti-mouse IgG-Cy3 (red) antibody to detect total AE1 expression. Cell images were examined using an LSM 510 confocal microscope. Colocalization (yellow) was calculated by Image J JACoP program using Pearson’s and Manders’s coefficients. Column A indicates cell surface mdAE1 (HA-Alexa 488 in green); column B indicates total mdAE1 (HA-cy3 in red); and column C indicates colocalization of cell surface mdAE1 and total mdAE1 (in yellow) on the cell surface. The average Pearson’s coefficients are WT 0.741, S725A 0.661, and S725R 0.419. TFIID, transcription factor IID.

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