Fig 2: Receptor-interacting protein kinases 4 (RIPK4) promotes pancreatic cancer tumor cell metastasis via the RAF1/MEK/ERK pathway. (A) Pathway enrichment analysis of differentially expressed genes using the KEGG pathway database. (B) The levels of total and phosphorylated RAF1, MEK1/2 and ERK1/2 in pancreatic cancer cell lines in which RIPK4 was overexpressed or knocked down. (C) The levels of SMAD2, p-SMAD2, GSK-3ß, p-GSK-3ß and p-ß-catenin in pancreatic cancer cell lines in which RIPK4 was overexpressed were detected by western blot analysis. (D and E) The effects of blocking RAF1/MEK/ERK signaling on pancreatic cancer cell (D) migration and (E) invasion were determined by Transwell assays using RIPK4-overexpressing Capan-1 and SW1990 cell lines. The numbers of migrating or invading cells were calculated, and the quantification of 3 randomly selected fields is shown in the histogram. *P<0.05.

Fig 3: Disruption of the KRXY motif accelerates X. laevis oocyte maturation. (A) pERK1/2 (44 kDa) immunoblotting of noninjected oocytes incubated at 5, 7, and 10 h with progesterone (10 µm); ERK2 (42 kDa) was used as a loading control (representative of three independent experiments). (B) Current traces recorded for the GIRK5-K13AR14A mutant. Voltage pulse protocols were performed using consecutive 100 ms step changes from -160 to +60 mV with increments of 20 mV. Oocytes were clamped at a holding potential of 0 mV. Silencing the gene encoding GIRK5 by specific siRNA suppressed K+ currents. (C) GIRK5 silencing was confirmed by EGFP-GIRK5K13AR14A (75 kDa) immunoblotting (representative of three independent experiments). (D) %GBVD was determined for noninjected and oocytes expressing: GIRK5 WT, GIRK5K13AR14A, and GIRK5K13AR14A plus siRNA GIRK5. Only GIRK5K13AR14A allowed progesterone-induced oocyte maturation up to 10 h. (*) Significant difference in oocyte maturation expressing WT or GIRK5K13AR14A compared to silenced GIRK5K13AR14A. (?) Significant difference in the maturation of oocytes expressing GIRK5K13AR14A at 5, 7 and 10 h compared to WT and noninjected oocytes (One-Way ANOVA, P < 0.01). Error bars correspond to mean ± SE, n = 15.

Fig 4: TNF70–80 activates p38 via the TNFRI. Neutrophils were treated with the indicated concentrations of either TNF or TNF70–80 and then incubated for 15 min before examining for p38 activation (a) or measuring chemiluminescence (b) over the 45-min period. The TNF70–80 induced chemiluminescence is inhibited by the p38 pharmacological inhibitor, SB203580. The neutrophils were pre-incubated with SB203580 for 10 min before the addition of TNF70–80. c TNF70–80 stimulates p38 activity in 70Z/3 pre-B cells, transfected with hTNFR1 (solid bars). Open bars represent mock-transfected cells. d Inhibition of biotin-labelled TNF70–80 binding to immobilised TNFR1 by unlabelled TNF70–80. e Peptide-TNFR1 binding was conducted as described under 'Methods'. Four-parameter curve fit yielded an IC50 value of 55 µM. (Inset) Lack of binding of scrambled peptide. The scrambled peptide and unlabelled TNF70–80 were both tested at 1 mM in the presence of 10 µM biotinylated TNF70–80. f TNF70–80 (10 µM) stimulated p38 activity in HEK 293T cells stably transfected with either wild-type TNF receptor-associated factor 2 (TRAF2) or an empty vector, but not in those transfected with a dominant-negative TRAF2 (?TRAF2). Results are mean ± s.e.m. of four experiments. Significance of difference (two-tailed Mann–Whitney U test): *p < 0.05; **p < 0.01, ***p < 0.001

Fig 5: Effect of caffeine on the TLR4/MAPK pathway of hepatic tissue from the NASH model. Protein levels of TLR4 (A), ERK (B), JNK (C), p38 (D), p-ERK (E), p-JNK (F), and p-p38 (G) in the hepatic tissues from rats in the control, HFSCD+CCl4, HFSCD+CCl4+caffeine, and caffeine groups were determined using western blot analysis. Bars represent the mean of experiments performed in duplicate ± SEM (n = 3); ß-actin was used as a loading control. p-ERK/ERK (H), p-JNK/JNK (I), and p-p38/p38 (J) ratios. Values are shown as a fold increment in optical density normalized to control group values (control = 1). Bars represent the mean of experiments performed in duplicate ± SEM. (a) p < 0.05 compared with the control group; (b) p < 0.05 compared with the HFSCD+CCl4 group.