Supplementary Figure 4: Y82C and I97M lead to TIM-3 protein misfolding and impaired surface expression. | Nature Genetics

Supplementary Figure 4: Y82C and I97M lead to TIM-3 protein misfolding and impaired surface expression.

From: Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome

Supplementary Figure 4

(a), Plot showing the impaired surface expression of the two TIM-3 mutants on the same HEK-293T cells as in Fig. 3d, using flow cytometry analysis, by APC-conjugated TIM-3 antibody or isotype control antibody. (b), Cell lysates from wild-type and Y82C or I97M TIM-3-transfected HEK-293 cells were put on a 7.5% native gel and immunoblotted against α-FLAG. Misfolded aggregates are highlighted with arrows. (c), The same lysates from (b) were submitted to an N-deglycosylation assay using the PNGase F enzyme and following the manufacturer’s denaturation protocol. These lysates as well as control lysates (without PNGase F) were immunoblotted with α-FLAG and α-β-actin. Experiments in (b) and (c) were repeated three times, and representative gels are shown. (d), Analysis of the wild-type and mutant (Y82C and I97M) bands before and after PNGase F treatment. MW, molecular weight.

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