American Society of Hematology

Fig. 1.

$Fig. 1. Expression of GMRβ N-glycosylation mutants in COS cell membranes. / Cells were transfected with expression plasmids encoding wild-type or mutated GMRβ. Proteins isolated from the membrane fractions of COS cells 48 hours after transfection were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with a rabbit antihuman GMRβ subunit antibody. (Panel A) One microgram of membrane protein from cells transfected with plasmids encoding wild-type or mutated GMRβ was loaded in each lane: 1, wild-type GMRβ; 2, GMRβ-Ala58; 3, GMRβ-Ala191; 4, GMRβ-Ala346; 5, mock transfectant. The immunoreactive bands were detected by enhanced chemiluminescence (ECL) with 1-minute exposure. (Panel B) Two micrograms of membrane protein from cells transfected with plasmids encoding wild-type or mutated GMRβ were loaded in each lane: 1, wild type GMRβ; 2, GMRβ-Asp58; 3, GMRβ-Asp191; 4, GMRβ-Asp346; 5, mock transfectant. The immunoreactive bands were detected by ECL with 1-minute exposure. (Panel C) One microgram of membrane protein obtained from cells transfected with plasmid encoding wild-type or GMRβ-Ala58/191/346 was loaded in each lane: 1, wild-type GMRβ without tunicamycin treatment; 2, wild-type GMRβ treated with 2.5 μg/mL tunicamycin; 3, GMRβ-Ala58/191/346. The immunoreactive bands were detected by ECL with 1-minute exposure. (Panel D) Twenty micrograms of membrane protein obtained from cells transfected with plasmid encoding GMRβ-Asp58/191/346 (lane 1) and mock transfectant (lane 2) were loaded. The immunoreactive bands were detected by ECL with 15 minutes of exposure.$

Expression of GMRβ N-glycosylation mutants in COS cell membranes.

Cells were transfected with expression plasmids encoding wild-type or mutated GMRβ. Proteins isolated from the membrane fractions of COS cells 48 hours after transfection were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with a rabbit antihuman GMRβ subunit antibody. (Panel A) One microgram of membrane protein from cells transfected with plasmids encoding wild-type or mutated GMRβ was loaded in each lane: 1, wild-type GMRβ; 2, GMRβ-Ala58; 3, GMRβ-Ala191; 4, GMRβ-Ala346; 5, mock transfectant. The immunoreactive bands were detected by enhanced chemiluminescence (ECL) with 1-minute exposure. (Panel B) Two micrograms of membrane protein from cells transfected with plasmids encoding wild-type or mutated GMRβ were loaded in each lane: 1, wild type GMRβ; 2, GMRβ-Asp58; 3, GMRβ-Asp191; 4, GMRβ-Asp346; 5, mock transfectant. The immunoreactive bands were detected by ECL with 1-minute exposure. (Panel C) One microgram of membrane protein obtained from cells transfected with plasmid encoding wild-type or GMRβ-Ala58/191/346 was loaded in each lane: 1, wild-type GMRβ without tunicamycin treatment; 2, wild-type GMRβ treated with 2.5 μg/mL tunicamycin; 3, GMRβ-Ala58/191/346. The immunoreactive bands were detected by ECL with 1-minute exposure. (Panel D) Twenty micrograms of membrane protein obtained from cells transfected with plasmid encoding GMRβ-Asp58/191/346 (lane 1) and mock transfectant (lane 2) were loaded. The immunoreactive bands were detected by ECL with 15 minutes of exposure.

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