Figure 3
Figure 3. Regulation of total and cell-surface HLA-E in cultured ECs upon inflammation. (A) HLA-E protein expression in untreated and IFNγ-activated ECs. Confluent monolayers of HAECs and HUVECs were incubated with culture medium or with 100 U/mL IFNγ for 48 hours. Cell lysates were immunoblotted using MEM-E/02 as anti–HLA-E mAbs. Immunoblots were reprobed with anti-GAPDH mAb to compare protein loading within samples. A representative immunoblot is shown. (B) Total-cell lysates from IFNγ-treated ECs were digested with EndoH or PNGaseF for 12 hours at 37°C, loaded onto a 10% SDS-PAGE, and examined by Western blotting with anti–HLA-E antibody. For controls, samples were incubated without the enzymes (NT). Immunoblots were reprobed with antitubulin mAbs. (C) FACS analysis showing cell-surface HLA-E expression (solid histograms) on HAECs and HUVECs either untreated or activated with IFNγ for 48 hours. Controls were performed by using an isotype-matched control antibody (empty histograms). Mean of fluorescence intensity are indicated. (D) Comparative analysis of HLA class Ia (HLA-A, HLA-B, and HLA-C) and HLA-E on the surface of 7 independently derived cultures of HAECs. Results are express as means of fluorescence intensity (MFI). Horizontal bars correspond to the means of value expressed as “mean of fluorescence intensity.” (E) Western blot analysis showing HLA-E, HLA-G, VCAM-1, and β2-microglobulin in cell lysates from HAECs treated for 48 hours with 100 U/mL TNFα, 2.5 ng/mL IL1β, or 100 U/mL IFNγ or culture medium alone (−). Immunoblots were reprobed with antitubulin mAbs to compare protein loading within samples. A representative immunoblot of 3 experiments is shown. (F) Western blot analysis showing HLA-E, classical HLA class I, VCAM-1, and β2-microglobulin in cell lysates from HAECs treated for 48 hours with 0 to 400 U/mL IFNγ. Immunoblots were reprobed with antitubulin mAbs to compare protein loading within samples. A representative immunoblot is shown. (G) Flow cytometry analysis comparing HLA-A2 and HLA-E expression at the cell surface after 48 hours of treatment with TNFα, IFNγ, IL-1β, TNFα plus IFNγ, or culture medium alone. MFIs are indicated.

Regulation of total and cell-surface HLA-E in cultured ECs upon inflammation. (A) HLA-E protein expression in untreated and IFNγ-activated ECs. Confluent monolayers of HAECs and HUVECs were incubated with culture medium or with 100 U/mL IFNγ for 48 hours. Cell lysates were immunoblotted using MEM-E/02 as anti–HLA-E mAbs. Immunoblots were reprobed with anti-GAPDH mAb to compare protein loading within samples. A representative immunoblot is shown. (B) Total-cell lysates from IFNγ-treated ECs were digested with EndoH or PNGaseF for 12 hours at 37°C, loaded onto a 10% SDS-PAGE, and examined by Western blotting with anti–HLA-E antibody. For controls, samples were incubated without the enzymes (NT). Immunoblots were reprobed with antitubulin mAbs. (C) FACS analysis showing cell-surface HLA-E expression (solid histograms) on HAECs and HUVECs either untreated or activated with IFNγ for 48 hours. Controls were performed by using an isotype-matched control antibody (empty histograms). Mean of fluorescence intensity are indicated. (D) Comparative analysis of HLA class Ia (HLA-A, HLA-B, and HLA-C) and HLA-E on the surface of 7 independently derived cultures of HAECs. Results are express as means of fluorescence intensity (MFI). Horizontal bars correspond to the means of value expressed as “mean of fluorescence intensity.” (E) Western blot analysis showing HLA-E, HLA-G, VCAM-1, and β2-microglobulin in cell lysates from HAECs treated for 48 hours with 100 U/mL TNFα, 2.5 ng/mL IL1β, or 100 U/mL IFNγ or culture medium alone (−). Immunoblots were reprobed with antitubulin mAbs to compare protein loading within samples. A representative immunoblot of 3 experiments is shown. (F) Western blot analysis showing HLA-E, classical HLA class I, VCAM-1, and β2-microglobulin in cell lysates from HAECs treated for 48 hours with 0 to 400 U/mL IFNγ. Immunoblots were reprobed with antitubulin mAbs to compare protein loading within samples. A representative immunoblot is shown. (G) Flow cytometry analysis comparing HLA-A2 and HLA-E expression at the cell surface after 48 hours of treatment with TNFα, IFNγ, IL-1β, TNFα plus IFNγ, or culture medium alone. MFIs are indicated.

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