Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: Co-immunoprecipitation (IP) of the Nef-ATG8 complex. Co-IP studies were carried out with lysates prepared from HEK293 cells stably expressing Nef-DsRed ( A–C ) and cotransfected with plasmids encoding for the indicated YFP-ATG8 constructs ( D–F ). ( A ) and ( D ) show samples before immunoprecipitation. In ( A ) an anti-GABARAPL2 (a), an anti-GABARAP (b), an anti-GABARAPL1 (c), and an anti-LC3B antibody (d) were used to detect the different ATG8s. ( B ) Immunoprecipitates with anti-DsRed antibody followed by SDS-PAGE and immunoblotting with various ATG8 antibodies (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B). ( C ) In a reciprocal set of experiments, immunoprecipitates were carried out with various ATG8 (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B) antibodies followed by SDS-PAGE and immunoblotting with an anti-DsRed antibody. In ( D ) an anti-DsRed antibody was used to detect Nef fused to DsRed and an anti-YFP antibody to detect YFP and the ATG8s fused to YFP. ( E ) Immunoprecipitates with an anti-DsRed antibody or ( F ) with an anti-GFP antibody followed by SDS-PAGE and immunoblotting with the antibodies are indicated. Data are representative of three independent experiments (U: unbound material; E: eluate fraction). Full-length western blots are presented in Supplementary Figs and .

Article Snippet: The HEK293 (Human embryonic kidney 293) cell line was obtained from DSMZ (Braunschweig, Germany) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM, GE Healthcare) with 4.5 g/l glucose containing 10% fetal bovine serum at 37 °C and in a 5% CO 2 atmosphere.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Construct, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: GABARAP, GABARAPL1 and GABARAPL2 colocalize with Nef in vesicular structures. ( A ) Live-cell fluorescence microscopy of HEK293 cells stably expressing Nef-CFP. Here, Nef was observed at cell-cell contact sites, bleb-like structures or at the plasma membrane (PM). Scale bar 20 µm. ( B ) Live-cell fluorescence microscopy of HEK293 cells transfected with peFP-C1/GABARAP (top), peYFP-C1/GABARAPL1 (middle) and peYFP-C1/GABARAPL2 (bottom) revealed a diffuse distribution pattern for all GABARAPs. Scale bar 20 µm. ( C ) Live-cell fluorescence microscopy of HEK293 cells stably expressing Nef-CFP (red) and transfected with peFP-C1/GABARAP (top), peYFP-C1/GABARAPL1 (middle) and peYFP-C1/GABARAPL2 (bottom) (all green). In this case, Nef and GABARAPs showed high colocalization levels at vesicular structures and at the plasma membrane (see merge and zoom). Scale bar 20 µm.

Article Snippet: The HEK293 (Human embryonic kidney 293) cell line was obtained from DSMZ (Braunschweig, Germany) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM, GE Healthcare) with 4.5 g/l glucose containing 10% fetal bovine serum at 37 °C and in a 5% CO 2 atmosphere.

Techniques: Fluorescence, Microscopy, Stable Transfection, Expressing, Clinical Proteomics, Membrane, Transfection

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: GABARAPs are required for plasma membrane localization of Nef. ( A ) Live-cell fluorescence microscopy of HEK293 cells stably expressing Nef-DsRed (red) and mock transfected as a control (top), transfected with scrambled siRNA (middle) or with a pool of GABARAP, -L1 and –L2 siRNAs (bottom). Hoechst 33442 was used to stain the nucleus (blue). Scale bar 20 µm. ( B ) Quantification of cells with Nef plasma membrane localization after treatment with the respective siRNAs from three independent experiments. ** p < 0.05. The average value of three control experiments was set to 100%.

Article Snippet: The HEK293 (Human embryonic kidney 293) cell line was obtained from DSMZ (Braunschweig, Germany) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM, GE Healthcare) with 4.5 g/l glucose containing 10% fetal bovine serum at 37 °C and in a 5% CO 2 atmosphere.

Techniques: Clinical Proteomics, Membrane, Fluorescence, Microscopy, Stable Transfection, Expressing, Transfection, Control, Staining