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. 2014 Jan;28(1):382–394. doi: 10.1096/fj.13-230037

Figure 6.

Figure 6.

Mfn2 is a Ras effector molecule. A) Sequence similarity between the C-terminal region of human Mfn2 (aa 490–757) and the C-terminal region of NORE1A (aa 191–413) containing RA and SARAH domains. Red box indicates the RA domain of NORE1A (aa 191–363); black box indicates the SARAH domain of NORE1A (aa 366–413). Asterisks indicate identity; colons indicate strong conservation; periods indicate weak conservation. B) Lysates (500 μg) from Mfn2−/− MEFs cotransfected with flag-tagged Mfn21–756 along with different HA-tagged K-Ras plasmids, WT-K-Ras, CA-K-Ras and CA-K-Ras-E37G, were immunoprecipitated with anti-HA monoclonal antibody (2 μg) and immunoblotted with either anti-Flag antibody (top panel) or anti-HA antibody (bottom panel). Total cell extracts (25 μg) were used to monitor the expression of flag-tagged Mfn2 and HA-tagged Ras. Similar results were obtained in 3 independent experiments. C) Expression levels of individual Ras constructs (WT-K-Ras, CA-K-Ras, and CA-K-Ras-E37G) were comparable. Mfn2−/− MEFs were cotransfected with empty vector and indicated HA-tagged Ras constructs, and 25 μg of total cell lysates was analyzed by WB analysis. D) Lysates (25 μg) from Mfn2−/− MEFs cotransfected with a flag-tagged Mfn2 plasmid together with H-Ras construct were analyzed by immunoblotting analysis. β-Tubulin was used as loading control. E) Total cell lysates (25 μg) from Mfn2−/− MEFs cotransfected with WT-Raf-1 together with indicated HA-tagged K-Ras constructs were analyzed by immunoblotting using anti-Raf-1 and anti-HA antibodies. β-Tubulin was used as loading control. F) Flag-tagged Mfn21-757 plasmid and indicated flag-tagged K-Ras constructs were expressed separately using in vitro transcription and translation system. Mfn2 protein was allowed to interact separately with the indicated Ras proteins first and then immunoprecipitated with anti-Ras antibody and immunoblotted with anti-Flag antibody. Similar results were obtained in 3 independent experiments. G, H) Mfn1 associates with Ras. Flag-tagged Mfn1 plasmid and HA-tagged CA-K-Ras plasmid were coexpressed in Mfn2−/− MEFs. Cell lysates (500 μg) were immunoprecipitated with either α-Flag monoclonal antibody (G) or α-HA monoclonal antibody (H), and the immunocomplexes were analyzed by WB analysis; 50 μg of total cell extracts was used as input.