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. 2015 Feb 17;112(9):2859–2864. doi: 10.1073/pnas.1414002112

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Fig. 3. — RNF121 facilitates ubiquitination and membrane localization of Nav1.6 in HEK293T cells. Protein extracts from cells transfected with RNF121_WT or RNF121_V228A, Na_V1.6-FLAG, and/or Na_Vβ₁-V5-His expression vectors. Proteasome activity was inhibited by MG132. Whole-cell extracts probed with anti-RNF121 (A), anti-FLAG (B), anti-His (C), or anti-GAPDH (D). Assessing the ubiquitination of Na_V1.6 from whole-cell extracts was achieved by immunoprecipitation with anti-FLAG, followed by probing with anti-FLAG (E) or anti-ubiquitin (F), which represents total and ubiquitinated Na_V1.6-FLAG, respectively. Membrane localization of Na_V1.6-FLAG and Na_Vβ₁-V5-His assayed through incubation of cells in biotin, followed by purification of biotinylated proteins and probing with anti-FLAG (G) or anti-His (H), respectively. Note that ubiquitination of Na_V1.6-FLAG was enhanced when coexpressed with RNF121_WT, and that membrane localization of Na_V1.6-FLAG increased when RNF121_WT and Na_Vβ₁-V5-His were coexpressed.