Integrity of the short arm of nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation

Published: 20-05-2021| Version 2 | DOI: 10.17632/8g59mp92bs.2
Contributor:
Valérie DOYE

Description

This dataset contains the original images for the six western blots presented in Gonzalez-Estevez, Verrico et al., 2021. Each folder corresponds to a given figure and encompasses a general montage, in which the areas used for the final Figure are indicated in red, as well as the original 16-bit images (for each antibody, the ECL signal and the image of the membrane to visualize the molecular markers are provided). The zip-file includes 6 folders corresponding to datasets (i): Figure 1B (Seh1-/-) (ii): Figure 5A (Mios-/-); Figure 5B (Seh1 Co-IP in Mios-/-); (iii) Figure 6B (Nup43-/-, ΔE2-GFP-Nup85); (iv) Figure S1D (GFP-mAID-Seh1) and (v) Figure S3C (ΔE2-GFP-Nup85, ΔE2-mCherry-Nup85, GFP-Seh1). Figure 1B: No Seh1 protein is detected in Seh1-/- clones and GFP-Seh1 migration in the Rescue clones (Seh1-/- mESCs stably expressing GFP-Seh1) is consistent with its predicted 71kDa molecular mass. The entire nitrocellulose membrane was first probed with Seh1, then cut and reprobed with Nup107 (Top) and GAPDH (bottom). Figure 5A: No Mios protein is detected in Mios-/- clones and Mios protein level is decreased in Seh1-/- samples compared to WT. The entire nitrocellulose membrane was first probed with Mios, then Seh1 and finally with Nup85. Figure 5B: Seh1 does not interact with Wdr24 in Mios-/- clones. Seh1 was Co-Immunoprecipitated (IP) in WT, Mios-/- and Seh1-/- mESC extracts, the latter being a negative control. The membrane was cut at 50kDa, and then probed with WDR24 (top, above 50 kDa) and Seh1 (below 50kDa). Figure 6B: No Nup43 protein is detected in Nup43-/- clones and ΔE2-GFP-Nup85 migration is consistent with its predicted molecular mass (98kDa). The entire nitrocellulose membrane was first probed with γ-Tubulin, then cut and reprobed with Nup85 (Top) and Nup43 and Seh1 (bottom). Figure S1D: GFP-mAID-Seh1 (predicted mass 75 kDa) is degraded upon auxin treatment. The entire nitrocellulose membrane was first probed with Seh1 antibody, then reprobed with gamma-Tubulin. Figure S3C: Characterization of the ΔE2-GFP-Nup85 and ΔE2-mCherry-Nup85 cell lines. The entire nitrocellulose membrane was first probed with Seh1, then cut and reprobed with Nup85 (Top) GAPDH and Nup43 (Bottom). GAPDH signal still present in Nup43 probe.

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