Supplementary Figure 5: Quality-control testing of the antibody to HLA-F used for immunoblot analysis and HLA-F mRNA probes for fluorescence in situ hybridization.
From: Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1
(a) Cell lysates (non-nuclear) from cell lines were run on reducing/denaturing SDS-PAGE, followed by immunoblotting (IB) for HLA-F and β-actin (loading control). Left image shows Jurkat cells (HLA-F−) and 721.221 cells (HLA-F+). Middle image shows THP-1 cells (HLA-F-deficient) and THP-1 cells transduced with N-terminally FLAG-tagged HLA-F. Right image shows Jurkat cells and Jurkat cells transduced with N-terminally FLAG-tagged HLA-F. (b) Fluorescent in situ hybridization and flow cytometry was done on an HLA-F+ cell line (BCL) and an HLA-F− cell line (Jurkat cells) with probes for HLA-F mRNA and also RPL13a mRNA (a housekeeping gene). Dotted histograms indicate staining without probes (‘– probe’; negative control) and solid-line, filled histograms are staining with probe (‘+ probe’).
