American Society of Hematology

Figure 5

$Figure 5. Biochemical properties of IFN-γR2 after various chemical treatments. (A) HEK293T cells were transiently transfected with 278delAG, WT, R114C, S124F, G141R, G227R, 382-387dup, and T168N IFNGR2-tagged V5 constructs. They were then incubated alone or with 166 μM kifunensine for 48 hours. Whole cell extracts were generated and left untreated or digested with Endo-H and PNGaseF overnight at 37°C. They were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibodies against V5 and glyceraldehyde-3-phosphate dehydrogenase. (B-C) Cell surface expression of IFN-γR2 was evaluated in a surface biotinylation assay in SV40 fibroblasts from the IFN-γR2–deficient patient (278delAG/278delAG) transfected with 278delAG, WT, S124F, R114C, G141R, G227R, 382-387dup, and T168N IFNGR2-tagged V5 constructs. These cells were then incubated alone or with 166 μM kifunensine for 48 hours. (B) Surface receptors isolated by precipitation with NeutroAvidin agarose beads. (C) Total extracts (T) or flow-through (F) fractions were immunoblotted with antibodies against V5, integrin β1, and glyceraldehyde-3-phosphate dehydrogenase.$

Biochemical properties of IFN-γR2 after various chemical treatments. (A) HEK293T cells were transiently transfected with 278delAG, WT, R114C, S124F, G141R, G227R, 382-387dup, and T168N IFNGR2-tagged V5 constructs. They were then incubated alone or with 166 μM kifunensine for 48 hours. Whole cell extracts were generated and left untreated or digested with Endo-H and PNGaseF overnight at 37°C. They were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibodies against V5 and glyceraldehyde-3-phosphate dehydrogenase. (B-C) Cell surface expression of IFN-γR2 was evaluated in a surface biotinylation assay in SV40 fibroblasts from the IFN-γR2–deficient patient (278delAG/278delAG) transfected with 278delAG, WT, S124F, R114C, G141R, G227R, 382-387dup, and T168N IFNGR2-tagged V5 constructs. These cells were then incubated alone or with 166 μM kifunensine for 48 hours. (B) Surface receptors isolated by precipitation with NeutroAvidin agarose beads. (C) Total extracts (T) or flow-through (F) fractions were immunoblotted with antibodies against V5, integrin β1, and glyceraldehyde-3-phosphate dehydrogenase.

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