Journal: The Journal of biological chemistry

Article Title: Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms.

doi: 10.1074/jbc.271.39.23775

Figure Lengend Snippet: FIG. 1. Activation of p38 MAPK by CDDP. NIH3T3 cells were treated with 50 mM CDDP for the indicated times (A) or with the indicated concentrations of CDDP for 3 h (B). Total cell lysates were immunoprecipitated with anti-p38 MAPK antibody, and in vitro immune complex kinase reactions were performed using GST-ATF2 fusion protein as substrate. The proteins were separated by 10% SDS-PAGE, stained with Coomassie Blue (second panels, A and B) and analyzed by autoradiography (top panels, A and B). Anti-p38 MAPK immunoprecipitates were also analyzed by immunoblotting with anti-p38 (third panels, A and B) or anti-Tyr(P) (fourth panels, A and B) antibodies. The fold increase in p38 MAPK activity is shown (bottom panels, A and B) as the mean (bars, S.E.) for three independent experiments.

Article Snippet: Lysates were incubated with anti-SAP kinase (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-p38 MAPK (16) antibodies for 1 h at 4 °C and then for 45 min after the addition of protein A-Sepharose.

Techniques: Activation Assay, Immunoprecipitation, In Vitro, Immune Complex Kinase Assay, SDS Page, Staining, Autoradiography, Western Blot, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms.

doi: 10.1074/jbc.271.39.23775

Figure Lengend Snippet: FIG. 2. Activation of p38 MAPK by different DNA-damaging agents in diverse cell types. A, U-937 cells were treated with 50 mM CDDP for 3 h. B, U-937 cells were treated with 50 mM ara-C for the indicated times. Total cell lysates were immunoprecipitated with anti- p38 MAP kinase antibody, and in vitro immune complex kinase reac- tions containing GST-ATF2 fusion protein were analyzed by 10% SDS-PAGE and autoradiography (upper panel). Anti-p38 MAPK immunoprecipitates from control and ara-C-treated cell lysates were also analyzed by immunoblotting with anti-Tyr(P) antibodies (lower panel). C, NIH3T3 cells were treated with 50 mM ara-C for the indicated times. The p38 MAPK activity was measured as described above. D, U-937 cells were treated with 20 gray IR and harvested at the indicated times. Total cell lysates were immunoprecipitated with either anti-p38 MAPK or anti-SAPK antibodies. In vitro immune complex kinase reac- tions containing GST-ATF2 (upper panel) or GST-Jun (bottom panel) fusion proteins were analyzed by 10% SDS-PAGE and autoradiography. E, NIH3T3 cells were treated with 50 mM CDDP for 3 h, 1 mM MMS for 3 h, or 80 J/m2 UV (harvested at 30 min). The p38 MAPK activity was measured as described above. The fold activation in kinase activities is shown at the bottom.

Article Snippet: Lysates were incubated with anti-SAP kinase (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-p38 MAPK (16) antibodies for 1 h at 4 °C and then for 45 min after the addition of protein A-Sepharose.

Techniques: Activation Assay, Immunoprecipitation, In Vitro, Immune Complex Kinase Assay, SDS Page, Autoradiography, Control, Western Blot, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms.

doi: 10.1074/jbc.271.39.23775

Figure Lengend Snippet: FIG. 3. c-Abl-dependent activation of p38 MAPK in response to CDDP. A, NIH3T3, Abl2/2 and Abl1 cells were treated with 100 mM CDDP for 3 h. Total cell lysates were immunoprecipitated with anti-p38 MAPK antibody, and in vitro immune complex kinase reactions con- taining GST-ATF2 fusion protein were analyzed by 10% SDS-PAGE and autoradiography (left panel). The fold increase in activity is shown at the bottom. Total cell lysates from NIH3T3, Abl2/2, and Abl1 cells were also immunoprecipitated with anti-Abl antibody. Protein precipi- tates were analyzed by immunoblotting with anti-Abl (right panel). B, Abl2/2 cells were treated with 100 mM CDDP for the indicated times. NIH3T3 cells were treated with 100 mM CDDP for 3 h as a positive control. p38 MAPK activity (upper panel) was assayed as described above. The fold increase in p38 MAPK activity is shown at the bottom panel (one of the three representative experiments is shown). C, NIH3T3 and Abl2/2 cells were treated with 50 mM ara-C for 3 h. Abl2/2

Article Snippet: Lysates were incubated with anti-SAP kinase (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-p38 MAPK (16) antibodies for 1 h at 4 °C and then for 45 min after the addition of protein A-Sepharose.

Techniques: Activation Assay, Immunoprecipitation, In Vitro, Immune Complex Kinase Assay, SDS Page, Autoradiography, Activity Assay, Western Blot, Positive Control

Journal: The Journal of biological chemistry

Article Title: Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms.

doi: 10.1074/jbc.271.39.23775

Figure Lengend Snippet: FIG. 4. Abl-dependent and -independent activation of p38 MAPK and SAP/JNK kinases by CDDP, MMS, or UV. NIH3T3 and Abl2/2 cells were treated with 1 mM MMS for 3 h (left panels), 100 mM CDDP for 3 h (left panels), or 80 J/m2 UV (harvested at 15 min) (right panels). Total cell lysates were immunoprecipitated with anti-p38 MAPK (A) or anti-SAPK (B) antibodies, and in vitro immune complex kinase reactions containing GST-ATF2 (A) or GST-Jun (B) fusion pro- teins were analyzed by 10% SDS-PAGE and autoradiography.

Article Snippet: Lysates were incubated with anti-SAP kinase (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-p38 MAPK (16) antibodies for 1 h at 4 °C and then for 45 min after the addition of protein A-Sepharose.

Techniques: Activation Assay, Immunoprecipitation, In Vitro, Immune Complex Kinase Assay, SDS Page, Autoradiography

Journal: European journal of immunology

Article Title: SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool.

doi: 10.1002/eji.202350788

Figure Lengend Snippet: Figure 4. Scarb1-deficiency affects the cholesterol concentration in the plasma membrane. (A) Representative histogram of unstimulated WT and Scarb1−/−BMMCs stained with the cholesterol- and GM1-specific probes GFP-D4 and CTB coupled to AlexaFluor647, respectively. The amount of cholesterol and GM1 within the extracellular leaflet of the PM of cells was assessed using both probes. (n = 5) (B) Signal intensities for GFP-D4 and CTB-AF647 quantified by MFI (n = 5) are depicted. (C) IgE-preloaded and starved WT BMMCs were pretreated with 10 mM MβCD for indicated time and subsequently stimulated or not with 20 ng/mL Ag (5 min). Cells were lysed and lysates were subjected to SDS-PAGE and Western blotting to analyze the activation of the PLCγ1 (Tyr783), PKB (Ser473), and MAPK pathways ERK1/2 (Thr202/Tyr204) and p38 (Thr180/Tyr183) by phospho-specific antibodies. Total proteins and phosphorylated proteins were detected on separate membranes, which are indicated by asterisks next to the protein name. Respective total protein blots served as loading controls. (n = 4) (D) Signal intensities from Western blot were analyzed by densitometry analysis of p-PLCγ1 and p-PKB in relation to the respective total protein amounts (n = 4). Data are shown as mean ± SD with individual data points representing independently performed experiments or as floating bars that indicate the range between the minimal and maximal values of the independent replicates while the line indicates the mean value. (B) was analyzed by multiple unpaired t-tests and corrected for multiple comparisons according to the Holm-Sídák method. (D) was analyzed by ordinary one-way ANOVA followed by the Dunnett test to correct for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The following antibodies were used for immunodetection of p-PLCγ1 (Tyr783, Cell Signaling Technology (CST), #2821), PLCγ1 (CST, #2822), p-PKB (Ser473, CST, #9271), PKB (CST, #9272), p-ERK1/2 (Thr202/Tyr204, CST, #4370), ERK1/2 (CST, #4696), p-p38 (Thr180/Tyr182, CST, #9216), p38 (Santa Cruz, #sc-81621), p-IκBα (Ser32, CST, #2859), IκBα (CST, #4812), p-KIT (Tyr719, CST, #3391), KIT (CST, #3074), SR-BI (Novus, #NB400-101), GAPDH (Santa Cruz, #166574), HSP 90 (CST, #4877).

Techniques: Concentration Assay, Clinical Proteomics, Membrane, Staining, SDS Page, Western Blot, Activation Assay

Journal: European journal of immunology

Article Title: SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool.

doi: 10.1002/eji.202350788

Figure Lengend Snippet: Figure 5. Cholesterol supports the synergistic response to SCF/Ag co-stimulation through PLCγ1 activation and the synergistic response of ROSAKIT WT cells is sensitive to SR-BI inhibition. (A) Representative Western blot of IgE-preloaded WT BMMC pretreated with either H2O or Chol. ox. (3 U/mL; 20 min) and subsequently left unstimulated or stimulated with Ag (20 ng/mL; 5 min), SCF (10 ng/mL; 10 min), or the combination of both stimuli (SCF 5 min prior to Ag for 5 min). Activation of signaling pathways was analyzed by Western blot using phospho-specific antibodies recognizing phosphorylation of PLCγ1 (Tyr783), PKB (Ser473), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr183). The respective amounts of detected total protein served as loading control. Total proteins and phosphorylated proteins were detected on separate membranes, which is indicated by an asterisk next to the protein name. GAPDH served as a control to prove equal loading of both membranes. (n = 3) (B) Quantification of the signal intensities of phosphorylated PLCγ1 and PKB relative to respective total protein in Western blot results from (A). Quantification was performed by densitometry analysis from n = 3 independent experiments. (C) Representative Western blot of IgE-preloaded WT BMMCs pretreated with DMSO as control or BLT-1 (3 μM) for 1 h and subsequently left unstimulated or stimulated with indicated concentrations of Ag for 5 min, 10 ng/mL SCF for 10 min or the combination (5 min SCF prestimulation followed by Ag stimulation for 5 min). Activation of signaling pathways was analyzed

Article Snippet: The following antibodies were used for immunodetection of p-PLCγ1 (Tyr783, Cell Signaling Technology (CST), #2821), PLCγ1 (CST, #2822), p-PKB (Ser473, CST, #9271), PKB (CST, #9272), p-ERK1/2 (Thr202/Tyr204, CST, #4370), ERK1/2 (CST, #4696), p-p38 (Thr180/Tyr182, CST, #9216), p38 (Santa Cruz, #sc-81621), p-IκBα (Ser32, CST, #2859), IκBα (CST, #4812), p-KIT (Tyr719, CST, #3391), KIT (CST, #3074), SR-BI (Novus, #NB400-101), GAPDH (Santa Cruz, #166574), HSP 90 (CST, #4877).

Techniques: Activation Assay, Inhibition, Western Blot, Protein-Protein interactions, Phospho-proteomics, Control

Journal: Journal of Integrative Agriculture

Article Title: Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

doi: 10.1016/s2095-3119(17)61670-8

Figure Lengend Snippet: Fig. 1 Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 4 (nsp4) interacts with cytochrome c1 (cyto.c1). A, yeast two hybrid assay. The plasmids pGBKT7-nsp4 and pGADT7-cyto.c1 were co-transformed into the yeast strain Y2HGold and selected on QDO/X/ABA plates. The top and second panels are the respective positive and negative controls. B, HEK 293T cells were transfected to express Flag-cyto.c1, or HA-nsp4 or together. At 24 h post-transfection, the cells were either subjected to direct Western blot analysis or lysed and immunoprecipitated with anti-HA antibodies. The proteins bound to sepharose beads were separated by SDS-PAGE, transferred to PVDF membranes, and probed with the antibodies to Flag and HA. C, the same as B except that nsp4 truncation mutants were used. D, MARC-145 cells were transduced with letiviruses that were expressing GFP or nsp4-GFP in the presence of 8 µg mL–1 polybrene, respectively. The cells were harvested 48 h post-transduction and cell lysates were immunoprecipitated using anti-GFP beads. The proteins bound to sepharose beads were subjected to Western blot analysis using an anti-cytochrome c1 polyclonal antibody or anti-GFP mAb. E, PRRSV nsp4 interacts with cyto.c1 in mammalian cells. Vero cells grown on coverslips in six-well plates were transfected when 60–70% to express the indicated proteins, either alone or pairwise. At 18–24 h post-transfection, the cells were fixed, permeablized, and stained with antibodies to HA and Flag, and examined by confocal microscopy. For double transfections, the merged images are shown at the right.

Article Snippet: Mouse anti-green fluorescent protein (GFP) mAb (66002-1-Ig) and rabbit anti-cytochrome c1 polyclonal antibodies (10242-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Virus, Y2H Assay, Transformation Assay, Transfection, Western Blot, Immunoprecipitation, SDS Page, Transduction, Expressing, Staining, Confocal Microscopy