Figure 3. Biotinylation of plasma... | Rockefeller University Press

Figure 3.

$Figure 3. Biotinylation of plasma membrane proteins. Kidneys were perfused with or without biotin. Lanes were loaded with 45 μg of total membrane protein or 25 μl of eluate from the neutravidin beads. Top: Na/Cl cotransporter (NCC). In total membranes, the antibody reacted with proteins of apparent molecular masses of ∼130 and ∼260 kD, presumably reflecting monomeric and dimeric forms of the transporter. The material in the biotinylated eluate was mostly at the higher molecular mass. No staining was observed in the nonbiotinylated eluate. Middle: Na/K/2Cl cotransporter (NKCC2). Three bands were observed, with the major ones at ∼140 and ∼280 kD, again presumably reflecting monomeric and dimeric forms of the transporter. The sharper band at ∼110 kD may represent the nonglycosylated form of the protein. The eluate of the biotinylated kidneys contained primarily the 280-kD form. No staining could be detected in the eluate from the nonbiotinylated kidneys. Bottom: Na/H exchanger (NHE3). The total membrane fraction contained a predominant band at ∼80 kD. The eluate from the biotinylated kidneys also contained this presumably full-length form of the protein, and also had a species at ∼160 kD that may represent a dimer. No staining could be detected in the eluate from the nonbiotinylated kidneys. (Panel has been rearranged so that the lanes correspond to those of the top two panels.)$

Biotinylation of plasma membrane proteins. Kidneys were perfused with or without biotin. Lanes were loaded with 45 μg of total membrane protein or 25 μl of eluate from the neutravidin beads. Top: Na/Cl cotransporter (NCC). In total membranes, the antibody reacted with proteins of apparent molecular masses of ∼130 and ∼260 kD, presumably reflecting monomeric and dimeric forms of the transporter. The material in the biotinylated eluate was mostly at the higher molecular mass. No staining was observed in the nonbiotinylated eluate. Middle: Na/K/2Cl cotransporter (NKCC2). Three bands were observed, with the major ones at ∼140 and ∼280 kD, again presumably reflecting monomeric and dimeric forms of the transporter. The sharper band at ∼110 kD may represent the nonglycosylated form of the protein. The eluate of the biotinylated kidneys contained primarily the 280-kD form. No staining could be detected in the eluate from the nonbiotinylated kidneys. Bottom: Na/H exchanger (NHE3). The total membrane fraction contained a predominant band at ∼80 kD. The eluate from the biotinylated kidneys also contained this presumably full-length form of the protein, and also had a species at ∼160 kD that may represent a dimer. No staining could be detected in the eluate from the nonbiotinylated kidneys. (Panel has been rearranged so that the lanes correspond to those of the top two panels.)

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