Fig 2: Potencies of ISJ towards the early response of major MAPK phosphorylation (in HepG2 and QGY-7703 cells) and for the late response of decreased invasion and apoptosis (in HepG2 cells).(a) ISJ inhibited the phosphorylation of c-Raf, MEK1/2 and ERK1/2 in the MAPK signaling pathway in both HepG2 and QGY-7703 cells. (b-c) ISJ treatment resulted in decreased invasion (b) and apoptosis (c) in HepG2 cells. GFX and GO were used as positive controls at final concentrations of 20 μM. *P < 0.05 versus the BLK controls. Data are shown as the mean ± SD of three independent experiments.

Fig 3: miR-202 expression blocks the activation of Raf-1/MEK/ERK signaling by suppressing K-Ras. ESCs were transfected with miR-202 mimics and pcDNA-k-Ras or co-transfected with both miR-202 mimics and pcDNA-K-Ras for 24 h before the cells were harvested for subsequent experiments. (A) Representative western blotting images of p-Raf1, Raf1, MEK, p-MEK, ERK1/2 and p-ERK1/2 protein levels. (B) The bands were semi-quantitatively analyzed using the Image J software, which were normalized to β-actin density. Data represent the mean ± SD from three independent experiments. *P<0.05 and **P<0.01 vs. Control, non-treated ESCs. ##P<0.01 vs. miR-202 mimics. p, phosphorylated; ESCs, endometrial stromal cells isolated from ectopic endometriosis tissues; miR, microRNA; NC, negative control.

Fig 4: The effects of cigarette smoke on phosphorylation of Raf-1 and ERK1/2 in rat coronary arteries.The coronary arterial segments were isolated from rats exposed to fresh air and cigarette smoke. The phosphorylated (p)-Raf-1 (A) and p-ERK1/2 (B) protein was shown relative to total Raf-1 or ERK1/2 level using Western blot (n = 6). Statistical analysis was performed using one-way ANOVA with Dunnett's post-test. * P<0.05 vs. fresh air group. (C) Immunofluorescence staining for phosphorylation of Raf-1 and ERK1/2. There was an increase in p-Raf-1 and p-ERK1/2 expression in the smooth muscle cell layer of coronary arteries from smoke exposed rats. Images were taken with a 40×objective.

Fig 5: Ras isoforms differ in their effector specificity and Ras isoforms exhibit a difference in symmetry in functional site surface roughness. a H-Ras, K-Ras and N-Ras specific siRNAs modulated the anti-CD40 antibody (3 μg/ml)-induced downstream Ras effector proteins Raf-1 and PI-3 K in P388D1 cells, as assessed by Western blot. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 10 min. b Analysis of association of H-Ras, K-Ras or N-Ras with PI3K or Raf in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 μg/ml and 6 μg/ml) treated macrophages for 10 mins. c The Correlation Dimensions (CD) were calculated to quantify the dependency in the spatial organization of surface atoms. CD magnitude varies within 2.00 and 3.00 for protein space. Results of this analysis too point unambiguously to similar organizational principles between dependencies (correlations) amongst surface atoms of H-Ras and K-Ras and their marked difference to that in N-Ras isoform. d Investigations were carried out on representative structures, viz., on pdb id.: 3K9L for H-Ras, pdb id.: 3GFT for K-Ras and pdb id.: 3CON for N-Ras. Extent of symmetry for all six aforementioned biophysical properties show that H-Ras and K-Ras molecules have a similar scheme of structural organization in interior and exterior of the molecule, which differs from that observed in N-Ras molecule. Such differences are significant because sequence identity between H-Ras, K-Ras and N-Ras molecules is found to be ≈ 89%.