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. 2012 Jul 23;7(7):e41891. doi: 10.1371/journal.pone.0041891

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Narita et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A and B. HEK293T cells were co-transfected with FLAG-wild-type-c-Myc together with two doses of HA-wild-type Rabring7 (A) or HA-C229S-Rabring7 (B). Forty-four hrs after transfection, 25 μM MG132 was added to the culture medium, and the cells were cultured for an additional 4 hrs. Proteins in cells were then immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with an anti-multi-ubiquitin antibody. C. HEK293T cells were co-transfected with FLAG-T58A-c-Myc together with two doses of HA-wild-type Rabring7 and subjected to ubiquitination assays as described in the legends of Figures 6A and 6B. D. HEK293T cells were co-transfected with FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Forty-eight hrs after transfection, total cell lysates were prepared and the cytoplasm and nucleus were fractionated as described in Experimental procedures. Proteins extracted from them were immunoprecipitated with an anti-FLAG antibody and analyzed by Western blotting with anti-HA, anti-FLAG and anti-T7 antibodies. Fractions of the cytoplasm and nucleus were also blotted with anti-HSP60 (SC-6216, Santa Cruz biotechnology, Santa Cruz, CA) and anti-Lamin B (SC-13115, Santa Cruz biotechnology) antibodies. E. HEK293T cells were co-transfected with EGFP-c-Myc, FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Proteins were analyzed as described in the legend of Figure 6D.