Journal:

Article Title: RIAM Regulates the Cytoskeletal Distribution and Activation of PLC-?1 in T cells

doi: 10.1126/scisignal.2000409

Figure Lengend Snippet: Knockdown of RIAM abrogates the translocation of RasGRP1 and CalDAG-GEFI to membranous fractions and impairs the activation of Ras and Rap1. RIAM was restored in RIAM-KD cells by transfecting them with a construct that encoded a variant RIAM that contained seven base mismatches with the sequence targeted by the RIAM-specific shRNA (RIAM-KD-R). (A) Control-KD, RIAM-KD, and RIAM-KD-R cells were stimulated with mAbs against CD3 and CD28 and the activation of Ras was examined by pull-down assays with a GST fusion protein of the Ras-binding domain (RBD) of Raf1. (B) The bar graph shows densitometric analysis of the abundance of Ras-GTP normalized to that of total Ras expressed as the fold-change relative to mean values prior to stimulation (defined as 1). Data are mean ±_SEM. Fold changes in the abundance of activated Ras were impaired in RIAM-KD cells. ( * p<0.05 versus control-KD; ♦ p<0.05 versus RIAM-KD-R; comparisons by ANOVA, n=3 for each cell type). (C) Whole-cell extracts from the same samples were analyzed for the activation of MEK1, MEK2, ERK1, ERJ2, JNK, and p38 by Western blotting with phospho-specific antibodies, followed by Western blotting with antiserum specific for ERK1 and ERK2 as a control. (D to G) The bar graphs show the densitometric analysis of changes in the abundance of phosphorylated substrates, normalized to ERK1/2 to control for loading variability and was expressed as a fold-change relative to mean values prior to stimulation (defined as 1). Comparable pp38 induction was observed in control-KD and RIAM-KD cells at all time interval. Significant reduction was observed in the induction of all other phosphorylated substrates at 5 and 15 minutes after stimulation in RIAM-KD cells (* p<0.05 versus control-KD; ♦ p<0.05 versus RIAM-KD-R; comparisons by ANOVA, n=4 for each cell type). (H) RIAM-KD, control-KD and RIAM-KD-R cells were stimulated with mAbs against CD3 and CD28 and membrane fractions were prepared as described before (4) and analyzed by Western blotting with antibodies against RasGRP1 and CalDAG-GEFI. The membrane-specific proteins calnexin, a marker of the endoplasmic reticulum (bottom panel), Na+/K+ ATPase, a marker of the plasma membrane (not shown), and p58, a marker of the Golgi (not shown) were used as controls of equal loading. (I, J) The bar graphs show the densitometric analysis of the translocation of RasGRP1 and CAlDAG-GEFI to the membrane. The abundance of RasGRP1 and CAlDAG-GEFI in the membrane fractions was normalized to that of calnexin to control for loading variability and expressed as the fold-change relative to mean values prior to stimulation (defined as 1). Fold changes in the abundance of membranous RasGRP1 and CALDAG-GEFI in control-KD cells compared to those in RIAM-KD and RIAM-KD-R cells were determined at each time point. Significantly reduced membrane translocation of RasGRP1 and CalDAG-GEFI was observed in RIAM-KD cells (* p<0.05 versus control-KD; ♦ p<0.05 versus RIAM-KD-R; comparisons by ANOVA, n=6 for each cell type). (K) The activation of Rap1 was examined by pull-down assays with GST-RalGDS-Ras binding domain (RBD) (L) The bar graph shows the densitometric analysis of the abundance of Rap1-GTP normalized to that of total Rap1, expressed as the fold-change relative to mean values prior to stimulation (defined as 1). Fold-changes in the abundance of activated Rap1 in control-KD cells compared to that in RIAM-KD and RIAM-KD-R cells were deteremined for each time point. Rap1 activation was reduced in RIAM-KD cells (* p<0.05 versus control-KD; ♦ p<0.05 versus RIAM-KD-R; comparisons by ANOVA, n=3 for each cell type).

Article Snippet: The following antibodies were used: RIAM polyclonal antibody #4612 (for immunoprecipitation), RIAM #5541 (for Western blotting) ( 10 ), and phosphotyrosine-specific mouse mAb (clone 4G10) were from Upstate Biotechnology; a mouse mAb against Ras was from Calbiochem; a rabbit mAb against Rap1 and a mouse mAb against CalDAG-GEFI were from Novus Bologicals; a mouse mAb against phosphotyrosine residues (clone 4G10), rabbit antiserum against Lck, a mouse mAb against ZAP-70, antiserum against Vav, and a mouse mAb against LAT were all from Upstate Biotechnology; a rabbit polyclonal antibody against Fyn, a goat polyclonal antibody against SLP-76, a mouse mAb against pERK, a mouse mAb against pJNK, a rabbit polyclonal antibody against RasGRP1, and goat antiserum against actin were from Santa Cruz Biotechnology; a mouse mAb against PLC-γ1 was from Abcam; rabbit polyclonal antibodies against pPLC-γ1 (Tyr 783 ) and pMEK1/2 (Ser 217/221 ) were from Cell Signaling Technology; a rabbit polyclonal antibody against pp38 (Thr 180 /Tyr 182 )was from New England Biolabs; a mouse mAB against the His tag was from Quiagen; rabbit antiserum against NFAT1 (S671.A20) has been previously described ( 46 ).

Techniques: Knockdown, Translocation Assay, Activation Assay, Construct, Variant Assay, Sequencing, shRNA, Control, Binding Assay, Western Blot, Membrane, Marker, Clinical Proteomics

Journal:

Article Title: Inhibition of mitochondrial permeability transition by cyclosporin A prevents pyrazole plus lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.freeradbiomed.2008.10.037

Figure Lengend Snippet: Levels of TNF-α, inducible nitric oxide synthase (iNOS) and activated p38 MAPK and JNK. (A) Western blot analysis of tumor necrosis factor (TNF)-α levels in liver homogenate subjected to 18% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Precursor of TNF-α (25 kD) is shown, *P<0.05, #P<0.01, vs. control mice. (B) Western blot analysis of iNOS levels in liver homogenate subjected to 8% SDS-PAGE. *P<0.05, vs. control mice. (C) Western blot analysis of phosphorylation of p38 MAPK. Liver homogenate subjected to 10% SDS-PAGE and immunoblotted with rabbit anti-p-p38 (Thy 180/Tyr 182) and p38 (H-147) antibodies as described in Materials and Methods. *P<0.05, vs. control mice. (D) Western blot analysis of c-Jun N-terminal kinase (JNK) with rabbit anti-JNK1 (C-17) and mouse anti-p-JNK (G7) antibodies. Anti-p-JNK (G7) antibody can react with phosphorylated JNK1, JNK2 and JNK3 at Thr-183 and Tyr-185. *P<0.005, vs. control mice. Numbers under the blot refer to the mean ± SD of the ratio of TNF-α, iNOS to β-actin, pp38 to p38, p-JNK2 to JNK2 from 3 mice in each group.

Article Snippet: Liver homogenate, 100 μg protein, was used for Western blot analysis of TNF-α (Rabbit anti-mouse TNF-α, 1 μg/ml, Fitzgerald Industries International Inc., Concord, MA), inducible nitric oxide synthase (iNOS) (Rabbit anti-iNOS, 1: 5000, Millipore Corp., Billerica, MA), phosphorylated and total p38 and JNK (Rabbit anti-pp38, p38, JNK1, and mouse anti-p-JNK antibodies, 1: 1000, Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page

Journal:

Article Title: Inhibition of mitochondrial permeability transition by cyclosporin A prevents pyrazole plus lipopolysaccharide-induced liver injury in mice

doi: 10.1016/j.freeradbiomed.2008.10.037

Figure Lengend Snippet: Scheme for the protection by cyclosporin A (CsA) against pyrazole plus lipopolysaccharide (LPS) liver injury. LPS induces Kupffer cells to release TNF-α, while pyrazole induces hepatocyte CYP2E1 expression. TNF-α plus CYP2E1 enhance production of ROS which can induce iNOS, and activate via phosphorylation p38 and JNK2 MAPK. Increases in ROS and reactive nitrogen species production, and activation of MAPK promotes an imbalance between anti-apoptic and pro-apoptotic BCL-2 family proteins, resulting in a mitochondrial permeability transition (MPT) and release of cytochrome c (Cyt c). This damage to the mitochondria increases production of ROS and causes hepatocyte necrosis. CsA prevents the MPT, blocks cytochrome c release from mitochondria, and lowers mitochondrial MDA, which protects the hepatocyte from the pyrazole plus LPS-induced necrosis.

Article Snippet: Liver homogenate, 100 μg protein, was used for Western blot analysis of TNF-α (Rabbit anti-mouse TNF-α, 1 μg/ml, Fitzgerald Industries International Inc., Concord, MA), inducible nitric oxide synthase (iNOS) (Rabbit anti-iNOS, 1: 5000, Millipore Corp., Billerica, MA), phosphorylated and total p38 and JNK (Rabbit anti-pp38, p38, JNK1, and mouse anti-p-JNK antibodies, 1: 1000, Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Activation Assay, Permeability

Journal: Journal of Neuroimmune Pharmacology

Article Title: Minocycline Abrogates Individual Differences in Nerve Injury-Evoked Affective Disturbances in Male Rats and Prevents Associated Supraspinal Neuroinflammation

doi: 10.1007/s11481-024-10132-y

Figure Lengend Snippet: Mean immunofluorescent intensity results for glial markers. ( A ) CD206 expression on IBA1 + microglia in combined bilateral hippocampal subfields and ( B ) in the ventroposterior lateral (VPL) thalamus in the contralateral (left) side as a percentage of the ipsilateral side. ( C ) CD206 expression on IBA1 + microglia in the ipsilateral and contralateral dorsolateral VPL thalamus. ( D ) Representative photomicrographs of the ventromedial VPL thalamus contralateral to the surgery site. ( E ) Representative photomicrographs (contralateral ventral pole CA1 of an affected rat) and fluorescent intensity values for BDNF staining on GFAP-positive cells, ( F ) IL-1β on IBA1 + cells, and ( G ) phospho-p38 MAPK on IBA1 + cells. Column graphs show group means ± standard error, with individual data points showing the expression value for one rat. Immunofluorescent intensity values are expressed in arbitrary units for mPFC and hippocampus, and as a percentage of the ipsilateral side for the aggregated VPL thalamus. CCI: chronic constriction injury; LD: unaffected ; HD: affected ; mPFC: medial prefrontal cortex; CD206: cluster of differentiation 206 (mannose receptor); BDNF: brain-derived neurotrophic factor; IL-1β: interleukin-1 beta; p38 MAPK: p38 mitogen-activated protein kinase. * P adj < 0.05; ** P adj < 0.01

Article Snippet: Sections were incubated with biotin- or Alexa series fluorophore-conjugated fab fragment secondary antibodies produced in donkey (Jackson ImmunoResearch) in 2% NHS in PBS for 3 h. FosB and pp38 MAPK underwent fluorophore-conjugated streptavidin amplification in PBS for 2 h. The primary antibody was then further incubated in 1:100 unconjugated fab fragments (Jackson ImmunoResearch, cat no. 711-007-003, RRID: AB_2340587) to block crossreactivity with the second primary antibody raised in the same species.

Techniques: Expressing, Staining, Derivative Assay

Journal: Stem Cell Reports

Article Title: RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells

doi: 10.1016/j.stemcr.2018.01.004

Figure Lengend Snippet: RAS Activity Is Induced in Primed ESCs (A–G) mESCs that were grown in 2i/LIF were switched to KSR/bFGF and grown for ten passages. Bright-field images showing cell morphology of each state (A). Real-time qPCR (B), immunofluorescent staining (C and D), and western blot (E) analyses were performed to examine the relative expression of the indicated markers of naive ( Stella and Klf4 ) or primed ( Fgf5 , Dnmt3b , and H3K9me3 ) state of pluripotency. DAPI is shown in (C) and (D). RAS-GTP pull-down assay was followed by western blot analysis using pan-RAS antibody (RAS-GTP) or using the indicated RAS isoform-specific antibodies (H-RAS-GTP, K-RAS-GTP, or N-RAS-GTP). The total expression of pan-RAS (RAS) or of each isoform (H-RAS, K-RAS, or N-RAS) was examined by western blot (F). The relative expression of active (phosphorylated) Ras-downstream effectors (pAKT, pERK, pJUN, and pP38) and the total expression was examined by western blot analysis using specific antibodies (G). (H and I) Real-time qPCR (H) and RAS-GTP pull-down assay (I), followed by western blot analysis using pan-RAS antibody (RAS-GTP), were performed in naive and primed hESCs. AKT was used as a loading control. (J) The levels of RAS-GTP, and total expression of pan-RAS and AKT (loading control), were examined in cell lysates from different mouse and human cell lines that were grown in 2i/LIF or in KSR/bFGF. mESC(1) = CGR8, mESC(2) = R1, hESC = H9, hiPSC(1) and hiPSC(2) were previously derived from skin fibroblast and hair follicles, respectively. (E–G, I, and J) Values represent densitometry analysis of three independent experiments. Scale bars, 100 μm in (A), 50 μm in (C), and 5 μm in (D). Data shown are mean ± SD from three independent experiments. ∗ p < 0.05 statistically significant by Student's t test.

Article Snippet: The membranes were probed with one of the following antibodies: mouse α-Pan-RAS (1:1,500, Calbiochem, OP40), mouse α-K-RAS (1:100, Calbiochem, OP24), mouse α-H-RAS (1:80, Calbiochem, OP23), mouse α-N-RAS (1:100, Calbiochem, OP25), rabbit α-ERK (1:3,500, Santa Cruz Biotechnology, sc154), mouse α-pERK (1:1,000, Abcam, ab201015), rabbit α-AKT (1:3,500, Cell Signaling Technology, no. 9272), rabbit α-pAKT (1:200, Cell Signaling Technology, no. 9271), rabbit α-pP38 (1:1,000, Abcam, ab195049), rabbit α-P38 (1:1,000, Cell Signaling Technology, sc7972), rabbit α-pJUN (1:1,000, Cell Signaling Technology, no. 9164), rabbit α-c-Jun (1:1,000, Santa Cruz Biotechnology, sc45), rabbit α-GFP (1:5,000, Abcam, ab13970), rabbit α-NANOG (1:1,000, PeproTech, 500-P236), rabbit α-H3K9me3 (1:1,000, Abcam, ab8898), rabbit α-DNMT3B (1:1,000, Abcam, ab2851), rabbit E-CAD (1:1,000, Santa Cruz Biotechnology, sc7870), rabbit α-N-CAD (1:1,000, Abcam, ab18203), and rabbit α-OCT3/4 (1:2,000, Santa Cruz Biotechnology, sc9081) at 4°C, overnight.

Techniques: Activity Assay, Staining, Western Blot, Expressing, Pull Down Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Sphingosine Kinase-1 (SphK-1) Regulates Mycobacterium smegmatis Infection in Macrophages

doi: 10.1371/journal.pone.0010657

Figure Lengend Snippet: WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS. Expression of LAMP-2 protein was monitored at 1 h and 4 h ( A ) and 24 h ( C ) post infection. The cells under sections ( A ) and ( C ) were also analyzed for the expression of pp38 at 1 h and 4 h ( A ) and iNOs at 1 h, 4 h ( B ) and 24 h ( C ) post infection time intervals. Shown here are the representative immunofluorescence pictures from two independent experiments. ( D ) Sphk-1 knockdown reduces the expression of major antimycobacterial proteins in macrophages. Both control and Sphk-1 siRNA knockdown macrophages were infected with M. smegmatis and expression of major antimycobacterial proteins was analyzed by western blot. Shown here is the representative blot from two independent experiments. ( E ) The blots were normalized against actin.

Article Snippet: Rabbit anti-mouse pp38 antibody was purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Infection, Expressing, Immunofluorescence, Knockdown, Control, Western Blot