Fig 1: Bone marrow and spleen from Gata1low mice contain levels greater than normal both of signaling elements downstream to TPO and of those downstream to TGF-β. (a, b) Western blot determinations of the JAK2, STAT5 and pSTAT5 content of bone marrow and spleen from wild-type and Gata1low mice (each lane). GAPDH was used as loading control. Quantification was obtained by normalizing each band to the corresponding GAPDH level and is presented as mean (±s.d.) with three mice per experimental group. P-values were determined by analysis of variance (ANOVA). (c, d) Western blot determinations of the SMAD2/3, phosphorylated p38 (p-p38), p38 and EZH2 content in the bone marrow and spleen from wild-type and Gata1low mice (each line a different mouse). GAPDH was used as a loading control. Quantification was obtained by normalizing p-p38 toward the corresponding p38 band, and SMAD2/3, p38 and EZH2 signals against GAPDH. P-values were calculated by ANOVA. EZH2, enhancer of zeste homolog 2.
Fig 2: Hydroxytyrosol, but not tyrosol, inhibits H2O2-induced sustained phosphorylation and activation of the JNK and p38 MAPKs. Jurkat cells (1.5×106 cells/ml) were exposed to 250 µm H2O2 for the indicated time points, in the absence or presence of 50 µm HTy (A) or Ty (B), added into the growth medium 30 min before the addition of H2O2. At the indicated time points phosphorylation levels of MAP kinases JNK, p38 and ERK were evaluated in total cell extracts by western blot analysis using specific antibodies. Quantification of band intensities for each MAPK is presented in graphs (lower panels) as the mean±SEM from three independent experiments, expressed as fold change relative to untreated cells (lines: black=H2O2; red=H2O2 plus HTy or Ty; blue=HTy or Ty alone)..
Fig 3: The Linearity tests for Quantifying Cellular Signaling Kinases. A series of negative (-) and positive (+) controls for phospho-specific and caspase 3 antibodies were mixed proportionally to generate artificial gradient of target proteins for the linearity tests using laser-amplified Qdot-RPPA. Examples shown here are (A) pAKT, (B) pERK, (C) pGSK3, (D) pNFkB, (E) pp38, (F) cleaved caspase 3. pAKT+, pERK+ and pGSK+ are Jurkat cells treated with phosphatase inhibitor, calyculin A, to accumulate these kinases in phosphorylated states. pNFkB+ is HeLa cells stimulated with TNF (20 ng/ml, 5 min) to activate NFkB. p-p38+ is C6 glioma cells treated with anisomycin to activate p38. Cleaved caspase 3+ is cytochrome c treated Jurkat cell lysates to induce the cleavage of caspase 3. X axis is the gradient (25% intervals) of targeted protein. Y axis is the calibrated signal intensity after subtracting the background signal at the 0% gradient. R2 values of linear regression curves close to 1 indicate laser-amplified Qdot-RPPA can distinguish at least 25% change of protein levels within the linear range. Error bars were derived from the three replicates of each sample.
Fig 4: Schematic representation of events taking place following exposure of cells to H2O2. in the presence of HTy. Exogenous H2O2 can penetrate easily the plasma membrane and reaches the cytosolic compartment (point 1). In cytosol, H2O2 is reduced to H2O by the enzymes GPx and Prx (point 2). However, when the intracellular concentration of H2O2 is elevated, it provokes the increase of cytosolic labile iron level (point 3), a fact that facilitates the sustained phosphorylation of MAP kinases JNK and p38 (point 4). H2O2 induces also the phosphorylation of ERK, but this event is iron independent (point 5). In the presence of iron-chelating compounds, like HTy, the labile iron level is reduced (point 6), thus, attenuating the H2O2-induced and labile iron-mediated prolonged phosphorylation of JNK and p38. Sustained phosphorylation of JNK and p38 leads ultimately to mitochondrial destabilization (point 7), followed by cytochrome C release, caspase 3 activation and cell apoptosis..
from Cell Signaling Technology for p38 MAPK Control Cell Extracts