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Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Supporting Information

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  • Download Appendix (PDF)
  • Download Dataset_S01 (XLSX)
  • Download Movie_S01 (AVI) - Synchronous activation of Xenopus laevis eggs with electroshock (first biological replicate). A field of unfertilized eggs from the frog Xenopus laevis undergoing activation that was induced by brief electrical shock. Each egg is at the single-cell stage with a diameter of 1.2-mm. The majority of eggs oriented with the pigmented animal pole (i.e., the top of the egg) facing upward. The white spot occurs from germinal vesicle breakdown during the first meiotic division. Before activation, these eggs are physiologically arrested in metaphase II. The movie shows the first ~7 min after electroshock, during which the cell cycle arrest is released and other events of the fertilization response are triggered (Fig. 1A). The synchrony of the activation is evident from two major morphological changes: (i) the cortical contractions, which can be seen as the “pulsing” of the pigmented area of the egg, and (ii) the more subtle calcium wave (a surface contraction wave), which visibly traverses the egg immediately before the cortical contractions. This second feature was used to quantify the synchrony of the activation in SI Appendix, Fig. S1. During the filming, sets of eggs were collected for proteomic analysis every 2 min (this occurred outside the field of view). See Materials and Methods for experimental details. The first biological replicate is labeled as “EA” (which stands for electroactivation) in all supporting documents. Images were taken every 5 s (frame rate of 14 frames/s).
  • Download Movie_S02 (AVI) - Synchronous activation of Xenopus laevis eggs with electroshock (second biological replicate). A field of unfertilized eggs from the frog Xenopus laevis undergoing activation that was induced by brief electrical shock. Each egg is at the single-cell stage with a diameter of 1.2-mm. The majority of eggs oriented with the pigmented animal pole (i.e., the top of the egg) facing upward. The white spot occurs from germinal vesicle breakdown during the first meiotic division. Before activation, these eggs are physiologically arrested in metaphase II. The movie shows the first ~7 min after electroshock, during which the cell cycle arrest is released and other events of the fertilization response are triggered (Fig. 1A). The synchrony of the activation is evident from two major morphological changes: (i) the cortical contractions, which can be seen as the “pulsing” of the pigmented area of the egg, and (ii) the more subtle calcium wave (a surface contraction wave), which visibly traverses the egg immediately before the cortical contractions (although this feature is more easily visualized in Movies S1, S3, and S4 as the pigmented regions are darker in those clutches). During the filming, sets of eggs were collected for proteomic analysis every 2 min (this occurred outside the field of view). See Materials and Methods for experimental details. The second biological replicate is labeled as “EAp2” (where “EA” stands for “electroactivation” and “p” denotes an experiment where phospho-enrichment occurred) in all supporting documents. Images were taken every 5 s (frame-rate of 14 frames/s).
  • Download Movie_S03 (AVI) - Synchronous activation of Xenopus laevis eggs with electroshock (third biological replicate). A field of unfertilized eggs from the frog Xenopus laevis undergoing activation that was induced by brief electrical shock. Each egg is at the single-cell stage with a diameter of 1.2-mm. The majority of eggs oriented with the pigmented animal pole (i.e., the top of the egg) facing upward. The white spot occurs from germinal vesicle breakdown during the first meiotic division. Before activation, these eggs are physiologically arrested in metaphase II. The movie shows the first ~7 min after electroshock, during which the cell cycle arrest is released and other events of the fertilization response are triggered (Fig. 1A). The synchrony of the activation is evident from two major morphological changes: (i) the cortical contractions, which can be seen as the “pulsing” of the pigmented area of the egg, and (ii) the more subtle calcium wave (a surface contraction wave), which visibly traverses the egg immediately before the cortical contractions. During the filming, sets of eggs were collected for proteomic analysis every 2 min (this occurred outside the field of view). See Materials and Methods for experimental details. The third biological replicate is labeled as “EApR3” (where “EA” stands for “electroactivation” and “p” denotes an experiment where phospho-enrichment occurred) in all supporting documents. Images were taken every 5 s (frame rate of 14 frames/s).
  • Download Movie_S04 (AVI) - Synchronous activation of Xenopus laevis eggs with electroshock (fourth biological replicate). A field of unfertilized eggs from the frog Xenopus laevis undergoing activation that was induced by brief electrical shock. Each egg is at the single-cell stage with a diameter of 1.2-mm. The majority of eggs oriented with the pigmented animal pole (i.e., the top of the egg) facing upward. The white spot occurs from germinal vesicle breakdown during the first meiotic division. Before activation, these eggs are physiologically arrested in metaphase II. The movie shows the first ~7 min after electroshock, during which the cell cycle arrest is released and other events of the fertilization response are triggered (Fig. 1A). The synchrony of the activation is evident from two major morphological changes: (i) the cortical contractions, which can be seen as the “pulsing” of the pigmented area of the egg, and (ii) the more subtle calcium wave (a surface contraction wave), which visibly traverses the egg immediately before the cortical contractions. During the filming, sets of eggs were collected for proteomic analysis every 2 min (this occurred outside the field of view). See Materials and Methods for experimental details. The fourth biological replicate is labeled as “EAp5b” (where “EA” stands for “electroactivation” and “p” denotes an experiment where phospho-enrichment occurred) in all supporting documents. Images were taken every 5 s (frame rate of 14 frames/s).

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