Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1 - Analysis of post-lysis interaction between PNGase-HA and Derlin-1-GFP. HeLa cells transfected separately with PNGase-HA and Derlin-1-GFP were lysed and cell lysates were mixed followed by immunoprecipitation with monoclonal anti-HA and reverse immunoprecipitation with monoclonal anti-GFP antibodies. Immunecomplexes captured on protein G agarose beads were resolved on SDS-PAGE and immunoblotted with antibodies as indicated. IP mono-HA; IB poly-HA (lane 1), IP mono-GFP; IB poly-HA (lane 2), IP mono-GFP; IB poly-GFP (lane 3) and IP mono-HA;IB poly-GFP (lane 4).
• Supplemental Figure 2 - Constructs of Derlin-1 used to study interaction and co-localization with PNGase.
• Supplemental Figure 3 - Co-localization of Derlin-1 constructs with PNGase-HA. HeLa cells were co-expressed with Derlin-1 constructs and PNGase-HA as indicated, stained with the corresponding antibodies, and visualized by fluorescence microscopy. A. Co-localization of Derlin-1-GFPΔ1-16 with PNGase-HA: Derlin-1-GFPΔ1-16 staining (green), PNGase-HA staining (red), merge shows the co-localization of Derlin-1GFPΔ1-16 with PNGase-HA. B. Co-localization of Derlin-1-GFPΔ1-154 with PNGase-HA: Derlin-1-GFPΔ1-154 staining (green), PNGase-HA staining (red), merge shows the co-localization of PNGase-HA and Derlin-1-GFPΔ1-154. C. Co-localization of Derlin-1-GFPΔ1-187 with PNGase-HA: Derlin-1-GFPΔ1-187 staining (green), PNGase-HA staining (red), merge shows the co-localization of Derlin-1-GFP Δ1-187 and PNGase-HA. D. Co-localization of Derlin-1-GFPΔ44-251 with PNGase-HA: Derlin-1-GFPΔ44-251 staining (green), PNGase-HA staining (red), merge shows the co-localization of Derlin-1-GFPΔ44-251 and PNGase-HA. E. Co-localization of Derlin-1-GFPΔ187-251 with PNGase-HA: Derlin-1-GFPΔ187-251 staining (green), PNGase-HA staining (red), merge shows the co-localization of Derlin-1-GFPΔ187-251 and PNGase-HA. DAPI (blue) shows nuclear staining.
• Supplemental Figure 4 - Black and White images showing the immunofluorescence localization of PNGase-HA and Derlin-1. HeLa cells were expressed with the plasmids as indicated, stained with the corresponding antibodies, and visualized by fluorescent microscopy. A. Co-localization of PNGase-HA with Derlin-1. B. Co-localization of PNGase-HA with calnexin. C. Co-localization of Derlin-1 with calnexin. D. Co-localization of pEGFP vector alone with PNGase-HA.