Fig. 4: Microtubule-associated proteome networks of Eml1 cKO neural progenitor cells.

A Overview of microtubule pelleting proteomic workflow for mouse neural progenitor cells derived from control and Eml1 cKO embryos. Pax6⁺ neural progenitor cells were isolated at E14.5, lysed, and treated with taxol to stabilize microtubules prior to pelleting. Pellet fractions were separated by SDS-PAGE and analyzed by LC-MS/MS. Cells were obtained from 4 WT and 4 Eml1 cKO embryos collected from 2 different litters, and each replicate was generated by pooling 2 embryos per genotype. Created in BioRender. Ozlu, N. (2025) https://BioRender.com/5nokslf. B Pelleted microtubule intensities of tubulin bands (50 kDa) from Coomassie-stained gels (Fig. 4A) were quantified using Fiji (ImageJ). Data represent mean ± SD from two biological replicates. Statistical significance was calculated using an unpaired two-tailed t-test; p = 0.046. C Among the 2805 quantified proteins, 347 (12.4%) were dysregulated in Eml1 cKO neural progenitor cells. D Volcano plot displays quantified proteins in microtubule pelleting assay, with the x-axis showing log₂ (Fold Change) Eml1 cKO/control and the y-axis representing −log₁₀(p-value) from the t-test. Within quantified proteins, unchanged proteins (gray), significantly downregulated proteins (blue), and significantly upregulated proteins (red) are labeled accordingly. Black vertical lines indicate the log₂ (Fold Change) threshold (±0.2), and the horizontal line marks the significance cutoff (p = 0.05). Tubulins, EMAPs, and Septins are labeled in the graph. E Western blot analysis of microtubule pelleting assay performed on mouse neural progenitor cells derived from control and Eml1 cKO embryos. Pax6⁺ neural progenitor cells were isolated at E14.5, lysed, and treated with either DMSO or taxol. Supernatant (S) and pellet (P) fractions were collected for analysis. Cells were obtained from 10 WT and 11 Eml1 cKO embryos collected from 3 different litters. Each replicate was generated by pooling at least 2 embryos per genotype. F Quantification of the % tubulin in the pellet fraction represented in (E). Error bars represent means ± SEM from three independent experiments. Statistical significance was calculated using one-way ANOVA; p < 0.0001 (****). G Sept2 levels in the pellet fraction, normalized to tubulin. Error bars represent means ± SEM from three independent experiments. Statistical significance was calculated using one-way ANOVA; p < 0.01 (**), p < 0.0001 (****). H STRING-db analysis of the dysregulated proteins highlighting the connection between Septins and EMAP family proteins. Lines between the proteins correspond to known interactions. Blue circles represent downregulated proteins in the microtubule pelleting of Eml1 cKO neural progenitor cells.