Fig 2: Protein levels in PBMCs of patients with non-syndromic autism and controls.a Left, representative Western blot showing protein levels of rpS6, eIF4E and p-eIF4E (Ser209). The molecular weight of each protein is indicated in kDa. Right, bar plots representing the quantification of the technical replicates where rpS6 and p-eIF4E levels are increased in ASD patients compared with controls (rpS6: U = 150, p = 0.03; p-eIF4E: U = 115, p < 0.0001). Error bars represent the standard error of the mean (n = 11-22 CTRL, 26-33 ASD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Mann–Whitney U tests). Each dot represents the average of at least two technical replicates per subject. Total proteins were normalised to the average of Coomassie staining and GAPDH. Phosphoproteins were normalised for respective total protein levels. *indicates the quantified signal b Left, representative Western blot showing protein levels of ERK 1-2, p-ERK 1-2 (Thr202/Tyr204), MNK1 and p-MNK1 (Thr197/202). The molecular weight of each protein is indicated in kDa. Right, bar plots representing the quantification of the technical replicates, where ERK1-2 and p-MNK1 levels are increased in ASD patients compared with controls (ERK1/2: U = 242.5, p = 0.04; p-MNK1: U = 58.5, p = 0.004)

Fig 3: Effects of ceranib-2 and SKI-II on rpS6, eIF4E, MNK1, and IKK in MV-infected PBL. In (A), Western blotting was performed to detect phosphorylated rpS6, total rpS6 protein, and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for phosphorylated over total rpS6 and total rpS6 protein over GAPDH signals are given. The increase of total rpS6 in comparison with the DMSO control was significant at 16 and 24 h after infection. The 16 and 24 h values of ceranib-2-treated infected cells and the values of 6, 16, and 24 h SKI-II-treated infected cells were significantly reduced in comparison with both, uninfected and infected cell values. The phosphorylation of rpS6 was significantly reduced by SKI-II at 16 and 24 h in comparison with infected cell values (n = 3; with *p ≤ 0.05). In (B), Western blotting was performed to detect phosphorylated eIF4E, total eIF4E protein, and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for phosphorylated over total eIF4E signals are given. Significances were calculated by student’s t-test by comparing corresponding time points of inhibitor treated with control signals (n = 3; with *p ≤ 0.05). In (C), Western blotting was performed to detect phosphorylated MNK1, total MNK1 protein, and GAPDH. Only the values for total MNK1 over GAPDH signals are given, because there were no significant differences in the phosphorylation of MNK1. Significances were calculated by student’s t-test by comparing corresponding time points of inhibitor treated with control signals (n = 3; with *p ≤ 0.05). In (D), Western blotting was performed to detect total IKK protein and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for total IKK over GAPDH signals are given. Significances were calculated by student’s t-test by comparing corresponding time points of inhibitor treated with control signals (n = 3; with *p ≤ 0.05). Some blots were used for detection of two different proteins (MNK1 and IKK) and GAPDH controls were re-used, and in some cases, two blots with lysates from the same experiment were used for the detection of total and phosphorylated proteins. The corresponding GAPDH controls were used for quantification. Only blots from one representative experiment are shown.

Fig 4: Schematic diagram depicting the genomic position of putative response elements on the (a) Mknk1 and (f) Mknk2 promoters relative to their TSS (chr4:115,511,826 and chr10:80,139,038, respectively). The sequences of the putative response elements are displayed in the tables below; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (b,h) P-CREB at putative CREs and (c,g,i,j) GR at putative GREs after 3 h of adipogenic induction. Enrichment was also assessed at the indicated NEG regions of the two genes (d,e,k,l). Data are mean ± SEM; n = 4; unpaired two-tailed t-test (*P < 0.05 **P < 0.01 ***P < 0.001)

Fig 5: Expressional differences in MNK/eIF4E signaling and molecular markers on EMT.(A) Protein expression of eIF4E, p-eIF4E, MNK1, and MNK2 in ccRCC and the normal kidney on an immunoblot. Cases 1, 2, and 3 belonged to different groups stratified with a combination of eIF4E with p-eIF4E IHC stainability in Figure 1F and 1G. (B) Relative transcript levels of MNKs in ccRCC and the normal kidney tissues. (C) Protein expression of eIF4E, p-eIF4E, MNK1, and EMT markers in cell lines on an immunoblot. (D) Relative levels of MNK1 and MNK2 mRNAs in HRCEpC and RCC cell lines. (E) mRNA levels of MNK2a and MNK2b in HRCEpC and RCC cell lines. The experiments were repeated at least three times. Representative data are shown. Error bars show standard deviations. Abbreviations. N: the normal kidney parenchyma, T: tumour, HRCEpC: human renal cortical epithelial cell.