Xenbase Image ID: 149835

Figure 4. Individual morpholinos specifically reduce 14-3-3 family member protein levels in Xenopus oocytes. A: Injection of 14-3-3 τ morpholino specifically reduced 14-3-3 τ protein level in Xenopus oocytes. Xenopus oocytes were injected with 20 or 40 pmol of 14-3-3 τ-specific morpholino or control morpholino. Protein lysates were generated after 72 hr and analyzed by anti–14-3-3 τ, 14-3-3 ζ, and total extracellular regulated kinase (ERK) immunoblotting. The 14-3-3 τ protein levels were decreased in specific morpholino-injected oocytes. The 14-3-3 ζ and total ERK levels served as loading controls. Data are representative of two separate 14-3-3 τ morpholinos. B: Injection of 14-3-3 ϵ morpholino specifically reduced 14-3-3 ϵ protein level in Xenopus oocytes. Both 14-3-3 ϵ morpholino designs (14-3-3 ϵ #1, 14-3-3 ϵ #2) reduced 14-3-3 ϵ protein level in Xenopus oocytes. Protein lysis and Western blotting were done as described above. C: The 14-3-3 β, γ, and ζ morpholinos were effective at reducing corresponding protein levels. A total of 20 to 40 pmol of specific morpholinos were injected into stage 6 Xenopus oocytes, and protein lysates were subjected to Western blotting as described above. D: The 14-3-3 family member-specific morpholinos were effective at blocking production of exogenously expressed 14-3-3 proteins in Xenopus oocytes. The 100 ng of myc-tagged 14-3-3 η, ϵ, or τ were injected into stage 6 oocytes and incubated at 19°C for 12 hr and protein lysate were subjected to Western blotting as described above. Each morpholino was effective at targeting the exogenously expressed 14-3-3 mRNAs. E: The 14-3-3 family member-specific morpholinos were effective at reducing corresponding protein levels in Xenopus embryos. Approximately 80–100 pmol of 14-3-3 β, τ, and control morpholinos were injected into two-cell stage embryos, incubated at room temperature for 24 hr after injection and protein lysates were subjected to Western blotting as described above. Western blotting showed the reduction of 14-3-3 τ in 14-3-3 τ morpholino-injected embryos, and reduction of 14-3-3 β in 14-3-3 β morpholino-injected embryos. Panels on the right show the quantification of relative 14-3-3 β and expressions when normalized to ERK loading. Image published in: Lau JM et al. (2006) Copyright © 2006. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View

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