Protocol for separation of the nuclear and the cytoplasmic fractions of Xenopus laevis embryonic cells for studying protein shuttling

Abstract

Summary This protocol for the separation of nuclear and cytoplasmic fractions of cells of Xenopus laevis embryos was developed to study changes in the intracellular localization of the Zyxin and Ybx1 proteins, which are capable of changing localization in response to certain stimuli. Western blot analysis allows the quantification of changes in the distribution of these proteins between the cytoplasm and nucleus, whereas the posttranslational modifications specific to each compartment can be identified by changes in electrophoretic mobility. For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020) .

Spatial and temporal proteomics reveals the distinct distributions and dynamics of O-GlcNAcylated proteins

“…Subsequently, cells were harvested and lysed with a buffer containing a low concentration of a mild detergent that preserved the nuclear envelope. The nuclear and cytoplasmic fractions were separated by centrifugation, as reported previously ( Martynova et al, 2021 ; Nabbi and Riabowol, 2015 ; Suzuki et al, 2010 ). To evaluate the separation of nuclear and cytoplasmic proteins, trypan blue staining and western blot experiments were performed.…”

The Cytoskeletal Protein Zyxin Inhibits Retinoic Acid Signaling by Destabilizing the Maternal mRNA of the RXRγ Nuclear Receptor

“…Xenopus laevis embryos were obtained with HCG injection as described by [ 28 ]. The embryos were staged as described by Nieuwkoop and Faber [ 29 ].…”

“…Embryos or AC were pretreated by incubation in 0.1 × MMR/1 × MMR containing 150 μg of cycloheximide per ml. After incubation for 1 h at 23 °C, nuclear and cytoplasmic fractions were prepared as described in [ 28 ].…”

Using RNA-binding proteins for immunoprecipitation of mRNAs from Xenopus laevis embryos

“… Timing: 45 min Collect embryos at the research stage (we work at the stage of middle gastrula, stage 11 according to the development table, Nieuwkoop table, see Figures 2 D and 2E and Martynova et al., 2021 , STAR Protocols: “The protocol for separation of the nuclear and the cytoplasmic fractions of Xenopus laevis embryonic cells for studying protein shuttling”). Place the embryos in Eppendorf tubes on ice, carefully remove the water, and add polysome lysis buffer (Buffers used for Immunoprecipitation of Ribonucleoprotein Complexes) with protease and RNase inhibitors at a rate of 10 μl of buffer per 1 embryo.…”

“…These can be detected by FLD fluorescence under a fluorescent microscope with a GFP filter. Dead embryos (easily visible as white spheres without dividing cells) non-FLD embryos are discarded (see Figure 2 F and Martynova et al., 2021 , STAR Protocols: “The protocol for separation of the nuclear and the cytoplasmic fractions of Xenopus laevis embryonic cells for studying protein shuttling”).…”