Figure 2. Proteomes and phosphoproteomes of wildtype and mutant embryos.

(A and C) Number of protein groups (A) or phosphosites (C) detected in wildtype, dorsalized (gd⁹), lateralized (Tl^rm9/Tlrm^rm10), and ventralized embryos (Toll^10B/def and spn27A^ex/def). (B and D) Intersection analysis of detected protein groups (B) or phosphosites (D). Black: detected in at least 1 replicate in all genotypes; green: detected in at least 1 replicate in all genotypes except 1 ventralized condition; white: detected in at least 1 replicate in any other combination. (E and F) Correlation matrix between the replicates of the proteomic (E) and phosphoproteomic (F) experiments using the Pearson correlation coefficient. Protein groups and phosphosites detected in all of the replicates in all of the genotypes were used to construct the correlation matrices. Proteomic (LFQ) analyses were performed using three technical replicates, with the exception of spn27a^ex/def and gd⁹ genotypes in which we used two biological replicates with three technical replicates each, making a total of six replicates for these two genotypes. For SILAC phosphoproteomic analyzes the protein lysate from embryos of each genotype was split in three and conducted three separate analyses. (G and H) Distribution of the number of protein groups (G) or phosphosites (H) exceeding an absolute fold change (vs. wild type, in log2 scale). Dotted line depicts the absolute fold change corresponding to 50% of the analyzed protein groups (G) or phosphosites (H).

Figure 2—figure supplement 1. Proteomic validation of dorso-ventral embryonic cell populations.

(A) log2 intensity (top) and log2 fold change (FC, bottom) of Toll and Spn27A proteins. (B) log2 intensity (top) and log2 fold change (bottom) of Toll phosphosite S871. (C) log2 intensity (top) and log2 fold change (bottom) of additional mesodermal proteins. (D) log2 intensity (top) and log2 fold change (bottom) of additional ectodermal fate determinants. (E) log2 intensity (top) and log2 fold change (bottom) of Cactus protein (left panels) and Cactus phosphosites (right panels): S463 (yellow dots), S467 (green dots), and S468 (magenta dots). Colors depict mutant genotypes and their comparisons against wild type: blue: dorsalized, magenta: lateralized, yellow: ventralized (Toll^10B/def and spn27A^ex/def). Bars depict mean and standard error of the mean across replicates. Absence of a dot indicates that the protein was not detected in a particular condition or that the log2 FC calculation was not feasible; absence of error bars in log2 intensity indicates that the protein was detected only in a single replicate. Dotted line indicates log2 FC = 0, log2 FC = 0.5 (for proteins) or log2 FC = 0.35 (for phosphosites). For all mean log2 intensity comparisons, we conducted a one-way ANOVA, followed by pairwise unpaired t-test comparisons (FDR corrected). Significance: * is p<0.05, ** is p<0.01, *** is p<0.001 and **** is p<0.0001. See Supplementary file 2 for ANOVA and pairwise comparison p-values.