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

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.

本数据集包含Gonzalez-Estevez、Verrico等人2021年发表的6张蛋白质免疫印迹（western blot）原始图像。每个文件夹对应一张指定图片，内含一张总拼接图——图中用于最终正式图片的区域以红色标注——同时还包含原始16位图像：针对每种抗体，分别提供了增强化学发光（enhanced chemiluminescence, ECL）信号图与用于可视化分子标记物的膜图像。
该压缩包共包含6个对应如下数据集的文件夹：(i) 图1B（Seh1基因敲除（Seh1-/-））；(ii) 图5A（Mios基因敲除（Mios-/-））、图5B（Mios基因敲除细胞中Seh1的免疫共沉淀（Co-Immunoprecipitation, Co-IP）实验）；(iii) 图6B（Nup43基因敲除（Nup43-/-）、ΔE2-GFP-Nup85）；(iv) 图S1D（GFP-mAID-Seh1）；(v) 图S3C（ΔE2-GFP-Nup85、ΔE2-mCherry-Nup85、GFP-Seh1）。
图1B：Seh1基因敲除（Seh1-/-）克隆中未检测到Seh1蛋白；挽救克隆（稳定表达GFP-Seh1的Seh1-/-小鼠胚胎干细胞（mouse embryonic stem cells, mESC））中GFP-Seh1的迁移速率与其预测的71kDa分子质量一致。整张硝酸纤维素膜（nitrocellulose membrane）首先用Seh1抗体孵育，随后将膜裁剪，上方片段用Nup107抗体重孵育，下方片段用GAPDH抗体重孵育。
图5A：Mios基因敲除（Mios-/-）克隆中未检测到Mios蛋白；与野生型（wild type, WT）样本相比，Seh1基因敲除（Seh1-/-）样本中的Mios蛋白水平显著降低。整张硝酸纤维素膜首先用Mios抗体孵育，随后依次用Seh1抗体与Nup85抗体进行重孵育。
图5B：Mios基因敲除（Mios-/-）克隆中Seh1无法与Wdr24发生相互作用。在野生型、Mios基因敲除与Seh1基因敲除小鼠胚胎干细胞裂解液中进行Seh1免疫共沉淀实验，其中Seh1基因敲除组作为阴性对照。将膜在50kDa分子量标记处裁剪，随后上方片段（分子量大于50kDa）用WDR24抗体孵育，下方片段（分子量小于50kDa）用Seh1抗体孵育。
图6B：Nup43基因敲除（Nup43-/-）克隆中未检测到Nup43蛋白；ΔE2-GFP-Nup85的迁移速率与其预测的98kDa分子质量一致。整张硝酸纤维素膜首先用γ-Tubulin抗体孵育，随后将膜裁剪，上方片段用Nup85抗体重孵育，下方片段用Nup43与Seh1抗体重孵育。
图S1D：GFP-mAID-Seh1（预测分子量75kDa）可在生长素（auxin）处理后发生降解。整张硝酸纤维素膜首先用Seh1抗体孵育，随后用γ-Tubulin抗体进行重孵育。
图S3C：ΔE2-GFP-Nup85与ΔE2-mCherry-Nup85细胞系的表征实验。整张硝酸纤维素膜首先用Seh1抗体孵育，随后将膜裁剪，上方片段用Nup85抗体重孵育，下方片段用GAPDH与Nup43抗体重孵育，其中Nup43检测组仍可观测到GAPDH信号。