Journal: Journal of Virology
Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking
Figure Lengend Snippet: M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.
Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).
Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Incubation, Purification, Staining, Immunofluorescence, Co-Immunoprecipitation Assay