Journal: Antioxidants (Basel, Switzerland)

Article Title: Preventive Effect of Probiotic Leuconostoc mesenteroides H40 Against Cognitive Disorder by Anti-Inflammatory, Synaptic Plasticity Regulation, and Antioxidant Effects.

doi: 10.3390/antiox14050565

Figure Lengend Snippet: Figure 6. Effects of probiotic Leuconostoc mesenteroides H40 on BDNF and CREP production. Gene expression of (A) BDNF and (B) CREB using rt-PCR. (C) BDNF/β-actin ratio and (D) p-CREB/CREB ratio using western blotting. Data are presented as the mean ± standard error. Different letters on the error bars indicate significant differences (n = 5, p < 0.05). NC, normal control without scopolamine; PC, positive control with scopolamine; DO, PC with 2 mg/kg donepezil; H40-7, PC with 1 × 107 CFU/mice·day H40; H40-8, PC with 1 × 108 CFU/mice·day H40; H40-9, PC with 1 × 109 CFU/mice·day H40. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the NC group. # p < 0.05 and ## p < 0.01 compared with the PC group.

Article Snippet: The nitrocellulose membranes were washed with distilled water and blocked with blocking buffer for 2 h. The membranes were then incubated for 12 h at 4 ◦C with antibodies specific to BDNF and CREB (Novus Biologicals, Centennial, CO, USA).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Positive Control

Journal: Frontiers in Behavioral Neuroscience

Article Title: Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice

doi: 10.3389/fnbeh.2016.00176

Figure Lengend Snippet: Hippocampal levels of phosphorylated cAMP response element-binding protein (pCREB) and CREB in the C57BL/6 and DBA/2 mice following the learning strategy-switching task. Representative immunoblots (A) of hippocampal pCREB and CREB from C57BL/6 and DBA/2 mice following the completion of the switched task and their quantification (B,C) . Hippocampal pCREB levels of C57BL/6 are higher than those of DBA/2 mice in both learning strategy-switching tasks (*, B ). Hippocampal CREB levels are lower in both C57BL/6 and DBA/2 mice receiving cued-to-place training compared to those receiving place-to-cued training (#, C ).

Article Snippet: Membranes were incubated with primary antibodies (Ab) against pCREB, phosphorylated on serine-133 (Millipore, #06-519) and total CREB (Cell Signaling, #9197).

Techniques: Binding Assay, Western Blot

Journal: Frontiers in Behavioral Neuroscience

Article Title: Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice

doi: 10.3389/fnbeh.2016.00176

Figure Lengend Snippet: Levels of pCREB and CREB in the lateral and medial regions of the dorsal striatum following the learning strategy-switching task. Representative immunoblots (A) of dorsal striatal pCREB and CREB from C57BL/6 and DBA/2 mice after the completion of the switched training task and their quantification (B,C) . Levels of pCREB in the medial region of the dorsal striatum are lower in C57BL/6 and DBA/2 mice receiving cued-to-place training compared to those receiving place-to-cued training (*, B ). Regional differences are not observed in pCREB levels but are detected in CREB levels (#, C ).

Article Snippet: Membranes were incubated with primary antibodies (Ab) against pCREB, phosphorylated on serine-133 (Millipore, #06-519) and total CREB (Cell Signaling, #9197).

Techniques: Western Blot

Journal: Frontiers in Behavioral Neuroscience

Article Title: Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice

doi: 10.3389/fnbeh.2016.00176

Figure Lengend Snippet: Levels of pCREB and CREB in the prefrontal cortex of the C57BL/6 and DBA/2 mice following the learning strategy-switching task. Representative immunoblots (A) of prefrontal cortex pCREB and CREB in C57BL/6 and DBA/2 mice (A) and their quantification (B,C) . After the cued-to-place training protocol, pCREB levels in the prefrontal cortex of C57BL/6 mice are significantly higher than those in DBA/2 mice (*, B ). CREB levels of C57BL/6 are higher than those of DBA/2 mice following place-to-cued training (#, C ).

Article Snippet: Membranes were incubated with primary antibodies (Ab) against pCREB, phosphorylated on serine-133 (Millipore, #06-519) and total CREB (Cell Signaling, #9197).

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

doi: 10.1523/jneurosci.0943-16.2017

Figure Lengend Snippet: Figure 1. Single-molecule imaging of CREB in cortical neurons. A, Experimental design of single-molecule imaging to visualize TMR-CREB in the nuclei of cortical neurons. A limited number of TMR-CREB molecules in the nucleus are observed with low background using HILO microscopy. B, A cortical neuron transfected with pT1-EGFP and HaloTag–CREB was observed at 7 DIV. C–K, Subcellular localization of TMR-CREB in fixed HaloTag–CREB-transfected cortical neurons using HILO fluorescence microscopy. C, Magnified image of the box in B. The cortical cells were immuno- stainedwithanti-laminA/Cantibody(G,H),anti-Ser5-RNAPIIC-terminaldomain(J,K),andCREBsignalswerevisualizedbyTMR-conjugatedHaloTagligand(D,E,F,H,I,K).I–K,Seriesofimages (0.2 m 50 sections) were subjected to a deconvolution algorithm. Arrows indicate TMR-CREB colocalized with Ser5-phosphorylated RNAP II C-terminal domain. Scale bar, 10 m. L–Q, The endogenousandexogenousCREBexpressionlevelintheHaloTag–CREB-expressingneurons.FluorescenceimagesofTMR-CREB(L–N)andimmunostainingwithanti-CREBantibody(O–Q)infixed HaloTag–CREB-transfected cortical neurons when adding different concentration of doxycycline (0, 0.05, 5 ng/ml). R, The quantitative analysis shows the relative expression level of CREB in transfected neurons treated with different doxycycline concentrations. Bars represent the mean SD. The number of neurons analyzed is 21 (0 ng/ml), 15 (0.05 ng/ml), and 15 (5 ng/ml) cells, respectively. The asterisk indicates a significant difference in untreated neurons (0 ng/ml; ANOVA with Tukey–Kramer post hoc tests, *p 0.001).

Article Snippet: The cells were then incubated overnight at 4°C with anti-lamin A/C mouse monoclonal antibody (39288, clone 3A6-4C11; Active Motif) at 1:1000, anti-CREB mouse monoclonal antibody (Cell Signaling Technology) at 1:200, anti-c-Fos rabbit monoclonal antibody (#2250; Cell Signaling Technology) at 1:100, or anti-RNAP II C-terminal domain phospho-Ser5 rabbit polyclonal antibody (Active Motif) at 1:10,000 in buffer G. Primary antibodies were detected by incubation with Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch) at 1:400 or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 in buffer G at room temperature for 2 h. Cells were mounted with a medium containing 50% glycerol and 2.3% 1,4-diazabicyclo[2.2.2 ]octane (Sigma-Aldrich), and 0.1% 4 ,6-diamidino-2-phenylimdole (DAPI, Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0.

Techniques: Imaging, Microscopy, Transfection, Fluorescence, Concentration Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

doi: 10.1523/jneurosci.0943-16.2017

Figure Lengend Snippet: Figure2. BindingdynamicsofCREBinthenucleioflivingcorticalneurons.A,HILOimageshowingindividualTMR-CREBspotsinthenucleusat5DIV.Scalebar,10m.B,Time-lapseimagesof theyellowboxinA.TheblueboxesindicateTMR-CREBspotswithlong(1s,spot1)andshort(1s,spot2)residencetimesatrestrictedlocations.Scalebar,2m.C,D,TMR-CREBspotsexhibit one-stepwise(C)ortwo-stepwise(D)reductionsofthefluorescenceintensities.Scalebar,0.5m.E,Thepiegraphshowstheratioofone-stepwiseandtwo-stepwisereductionofthefluorescence intensities in wild-type and mutant CREB (L318/325V, R301L).

Article Snippet: The cells were then incubated overnight at 4°C with anti-lamin A/C mouse monoclonal antibody (39288, clone 3A6-4C11; Active Motif) at 1:1000, anti-CREB mouse monoclonal antibody (Cell Signaling Technology) at 1:200, anti-c-Fos rabbit monoclonal antibody (#2250; Cell Signaling Technology) at 1:100, or anti-RNAP II C-terminal domain phospho-Ser5 rabbit polyclonal antibody (Active Motif) at 1:10,000 in buffer G. Primary antibodies were detected by incubation with Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch) at 1:400 or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 in buffer G at room temperature for 2 h. Cells were mounted with a medium containing 50% glycerol and 2.3% 1,4-diazabicyclo[2.2.2 ]octane (Sigma-Aldrich), and 0.1% 4 ,6-diamidino-2-phenylimdole (DAPI, Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0.

Techniques: Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

doi: 10.1523/jneurosci.0943-16.2017

Figure Lengend Snippet: Figure3. Thebehaviorofwild-typeCREBcomparedwithtwomutantCREBs.A,B,Distribu- tion histograms show the short (1 s; A) and long (1 s; B) residence times of TMR-CREB, TMR-mutantCREB(R301L),andTMR-mutantCREB(L318/325V)inlivingneurons.Thenumber of spots analyzed in living cells is presented in parentheses.

Article Snippet: The cells were then incubated overnight at 4°C with anti-lamin A/C mouse monoclonal antibody (39288, clone 3A6-4C11; Active Motif) at 1:1000, anti-CREB mouse monoclonal antibody (Cell Signaling Technology) at 1:200, anti-c-Fos rabbit monoclonal antibody (#2250; Cell Signaling Technology) at 1:100, or anti-RNAP II C-terminal domain phospho-Ser5 rabbit polyclonal antibody (Active Motif) at 1:10,000 in buffer G. Primary antibodies were detected by incubation with Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch) at 1:400 or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 in buffer G at room temperature for 2 h. Cells were mounted with a medium containing 50% glycerol and 2.3% 1,4-diazabicyclo[2.2.2 ]octane (Sigma-Aldrich), and 0.1% 4 ,6-diamidino-2-phenylimdole (DAPI, Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

doi: 10.1523/jneurosci.0943-16.2017

Figure Lengend Snippet: Figure 4. Dynamics of CREB in response to changing neuronal activity by pharmacological treatment. A, Subcellular localization of TMR-CRTC1 in fixed HaloTag–CRTC1-transfected neu- rons with or without KCl treatment (50 mM) for 1 h at 7 DIV. Immunocytochemical staining wasalsoperformedwithc-fosantibodyandDAPI.Scalebar,10m.B,Distributionhistograms showthelong(1s)residencetimesofTMR-CREBspotsinuntreated(control)corticalneurons at7DIVorinthosetreatedwith50mMKClfor1h.Thenumberofspotsanalyzedispresentedin parentheses. C, Distribution histograms show the long (1 s) residence times of TMR-CREB spotsinuntreated(control)corticalneuronsat14DIVandinthosetreatedwithTTXfor24h.The number of spots analyzed is presented in parentheses.

Article Snippet: The cells were then incubated overnight at 4°C with anti-lamin A/C mouse monoclonal antibody (39288, clone 3A6-4C11; Active Motif) at 1:1000, anti-CREB mouse monoclonal antibody (Cell Signaling Technology) at 1:200, anti-c-Fos rabbit monoclonal antibody (#2250; Cell Signaling Technology) at 1:100, or anti-RNAP II C-terminal domain phospho-Ser5 rabbit polyclonal antibody (Active Motif) at 1:10,000 in buffer G. Primary antibodies were detected by incubation with Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch) at 1:400 or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 in buffer G at room temperature for 2 h. Cells were mounted with a medium containing 50% glycerol and 2.3% 1,4-diazabicyclo[2.2.2 ]octane (Sigma-Aldrich), and 0.1% 4 ,6-diamidino-2-phenylimdole (DAPI, Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0.

Techniques: Activity Assay, Transfection, Staining, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

doi: 10.1523/jneurosci.0943-16.2017

Figure Lengend Snippet: Figure 5. CREB behavior in response to stimulation using ChR2 optogenetics. A, Subcellular localization of TMR-CRTC1 was observed by time-lapse imaging after the onset of photostimulation (0.1 Hz, 1 s duration) in an EYFP-expressing or ChR2-EYFP- expressingcorticalneuronat5DIV.Representativeimagesat0,200,400,and600sareshown.Scalebar,10m.B,Fluorescence intensities of TMR-CRTC1 in the nucleus of an EYFP-transfected or ChR2-EYFP-transfected cortical neuron were analyzed in the seriesofimagesrepresentedinA.C,SubcellularlocalizationofTMR-CRTC1wasobservedbeforeandafterphotostimulation(2Hz, 50msduration)for10mininaChR2-EYFP-expressingcorticalneuronat5DIV.Scalebar,10m.D,ATMR-CREB-expressingand ChR2-EYFP-expressing cortical neurons. Scale bar, 10 m. E, F, Distribution histograms show the long (1 s) residence times of TMR-CREB spots in cotransfected neurons at 5 DIV, before (pre) and after (post) photostimulation (E: 0.1 Hz, 1 s duration; F: 2 Hz, 50msduration)for5min.G,Distributionhistogramsshowthelong(1s)residencetimesofTMR-mutantCREB(R301L)spotsin cotransfectedneuronsat5DIV,before(pre)andafter(post)photostimulation(0.1Hz,1sduration)for5min.Thenumberofspots analyzed is presented in parentheses.

Article Snippet: The cells were then incubated overnight at 4°C with anti-lamin A/C mouse monoclonal antibody (39288, clone 3A6-4C11; Active Motif) at 1:1000, anti-CREB mouse monoclonal antibody (Cell Signaling Technology) at 1:200, anti-c-Fos rabbit monoclonal antibody (#2250; Cell Signaling Technology) at 1:100, or anti-RNAP II C-terminal domain phospho-Ser5 rabbit polyclonal antibody (Active Motif) at 1:10,000 in buffer G. Primary antibodies were detected by incubation with Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch) at 1:400 or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 in buffer G at room temperature for 2 h. Cells were mounted with a medium containing 50% glycerol and 2.3% 1,4-diazabicyclo[2.2.2 ]octane (Sigma-Aldrich), and 0.1% 4 ,6-diamidino-2-phenylimdole (DAPI, Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0.

Techniques: Optogenetics, Imaging, Expressing, Fluorescence, Transfection

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging

doi: 10.1523/jneurosci.0943-16.2017

Figure Lengend Snippet: Figure 6. Dynamics of CREB hot spots in the nuclei of living cortical neurons. A–C, The number of TMR-CREB spots for the entire observation time (60 s) was counted in each subarea. For the nucleusdepictedhere,eightand11of157spots(5%)accumulatedinthetwodifferentsubnuclearareasdefinedashotspots(red),indicatedbythearrows.A,Theareacoveringthewholenucleus in a cortical neuron was divided into 625 (25 25) equal subareas (0.8 0.8 m). Scale bar, 10 m. Color contour maps represent the spatial distribution of the frequency of appearance of TMR-CREBspotshavinglongresidencetimes(1s)inthelivingneuronshowninAbefore(pre,B)andafter(post,C)photostimulation.Thedashedlinemarkstheperimeterofthenucleus.D,The distributionhistogramshowsthenumberofhotspotsbefore(pre)andafter(post)photostimulation(0.1Hz,1sduration;n 26cells).Theaveragenumberofhotspotswassignificantlydifferent before (0.1 0.1) and after stimulation (1.4 0.2; Mann–Whitney U test, p 0.001). E, The distribution histogram shows the number of hot spots of TMR-CREB before (pre) and after (post) photostimulation(2Hz,50msduration;n 19cells)for5min.Theaveragenumberofhotspotswassignificantlydifferentbefore(0.10.1)andafterstimulation(1.60.4;Mann–WhitneyU test, p 0.001). F, The histogram shows the number of TMR-CREB spots with long-residence times (1 s) observed in hot spots (n 37) or other sites (n 32) in 26 cells. Bars represent the meanSEM.Theasteriskindicatesasignificantdifferencebetweenbefore(pre)andafter(post)photostimulation(Mann–WhitneyUtest,*p0.001).G,Thedistributionhistogramshowsthe numberofhotspotsintwosuccessivecontrolmeasurementswithoutphotostimulation(n 16cells).Theaveragenumberofhotspotsinthefirst(1st;0.10.1)andthesecond(2nd;0.40.2) measurementwasnotsignificantlydifferent(Mann–WhitneyUtest,p 0.686).H,ThedistributionhistogramshowsthenumberofhotspotsofmutantCREB(R301L)before(pre)andafter(post) photostimulation(0.1Hz,1sduration;n 6cells)for5min.Theaveragenumberofhotspotswasnotsignificantlydifferentbefore(0.20.2)andafterstimulation(0.20.2;Mann–Whitney U test, p 1.000).

Article Snippet: The cells were then incubated overnight at 4°C with anti-lamin A/C mouse monoclonal antibody (39288, clone 3A6-4C11; Active Motif) at 1:1000, anti-CREB mouse monoclonal antibody (Cell Signaling Technology) at 1:200, anti-c-Fos rabbit monoclonal antibody (#2250; Cell Signaling Technology) at 1:100, or anti-RNAP II C-terminal domain phospho-Ser5 rabbit polyclonal antibody (Active Motif) at 1:10,000 in buffer G. Primary antibodies were detected by incubation with Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch) at 1:400 or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 in buffer G at room temperature for 2 h. Cells were mounted with a medium containing 50% glycerol and 2.3% 1,4-diazabicyclo[2.2.2 ]octane (Sigma-Aldrich), and 0.1% 4 ,6-diamidino-2-phenylimdole (DAPI, Sigma-Aldrich) in 50 mM Tris-HCl, pH 8.0.

Techniques: MANN-WHITNEY

Journal: Oncogene

Article Title: Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells

doi: 10.1038/s41388-019-0843-1

Figure Lengend Snippet: A-B) p-IGF1R, p-STAT3, and DARPP-32 protein levels were determined by Western blot analysis in AGS cells with stable expression of DARPP-32 (DP32) or MKN45 cells with siRNA knockdown of DARPP-32. C-D) Luciferase reporter assay for STAT3-luc following siRNA knockdown of IGF1R in cells with stable expression of DARPP-32 (DP32) or DARPP-32 siRNA knockdown. Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test.

Article Snippet: Immunofluorescence was performed following cell fixation and permeabilization, using antibodies against DARPP-32 (1:400) (Santa Cruz, sc-398144), p-STAT3 (Cell Signaling, Y705, 9193s) or p-SRC (1:400) (Cell Signaling, Y416, 6943s).

Techniques: Western Blot, Expressing, Knockdown, Luciferase, Reporter Assay

Journal: Oncogene

Article Title: Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells

doi: 10.1038/s41388-019-0843-1

Figure Lengend Snippet: A) Western blots analysis of IGF1R, p-IGF1R, SRC, and p-SRC in AGS cells stably expressing DARPP-32 (DP32), following IGF1R siRNA knockdown. B) p-IGF1R, SRC, and p-SRC protein levels were determined by Western blot analysis in MKN45 cells, following DARPP-32 siRNA knockdown and treatment with OSI-096 (2 μg/ml) or vehicle. C-D) Luciferase reporter assay for STAT3-luc following treatment with OSI-096 (2 μg/ml) in AGS cells stably expressing DARPP-32 (DP32), using pcDNA-DARPP-32 or DARPP-32 siRNA knockdown in MKN45 cells. Statistical significance in all panels was calculated by 1-way ANOVA, followed by the student’s t test.

Article Snippet: Immunofluorescence was performed following cell fixation and permeabilization, using antibodies against DARPP-32 (1:400) (Santa Cruz, sc-398144), p-STAT3 (Cell Signaling, Y705, 9193s) or p-SRC (1:400) (Cell Signaling, Y416, 6943s).

Techniques: Western Blot, Stable Transfection, Expressing, Knockdown, Luciferase, Reporter Assay

Journal: Oncogene

Article Title: Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells

doi: 10.1038/s41388-019-0843-1

Figure Lengend Snippet: A) Western blots analysis of p-SRC, p-STAT3, and DARPP-32 in AGS cells stably expressing DARPP-32 (DP32), using pcDNA-DARPP-32, following knockdown of SRC by SRC siRNA. B) p-SRC, p-STAT3, and DARPP-32 protein levels were determined by Western blot analysis in AGS cells stably expressing DARPP-32 (DP32), following treatment with Dasatinib (10 μM) or vehicle. C) Luciferase reporter assays for STAT3-luc were performed following treatment with Dasatinib (10 μM) in AGS cells stably expressing DARPP-32 (DP32). Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test. D) p-SRC, p-STAT3, and DARPP-32 protein levels were determined by Western blot analysis in DARPP-32 siRNA knockdown MKN45 cells following treatment with Dasatinib (10 μM) or vehicle. E) Luciferase reporter assay for STAT3-luc were performed following Dasatinib (10 μM) treatment in DARPP-32 siRNA knockdown MKN45 cells. Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test. F) Immunofluorescence analysis using AGS cells, following overexpression of DARPP-32 (red), demonstrates an increase in expression of p-SRC (green) in DARPP-32-expressing AGS cells (left panel). Immunofluorescence analysis in MKN45 cells, following DARPP-32 siRNA knockdown, demonstrates a decrease of p-SRC in DARPP-32 siRNA/MKN45 cells; DARPP-32 (red), p-STAT3 (green) (right panel).

Article Snippet: Immunofluorescence was performed following cell fixation and permeabilization, using antibodies against DARPP-32 (1:400) (Santa Cruz, sc-398144), p-STAT3 (Cell Signaling, Y705, 9193s) or p-SRC (1:400) (Cell Signaling, Y416, 6943s).

Techniques: Western Blot, Stable Transfection, Expressing, Knockdown, Luciferase, Reporter Assay, Immunofluorescence, Over Expression

Journal: Oncogene

Article Title: Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells

doi: 10.1038/s41388-019-0843-1

Figure Lengend Snippet: A) Immunofluorescence analysis of p-STAT3 (green) and DARPP-32 (red) in gastric organoids established from wild-type, TFF1 KO, DP KO and TFF1 KO/DP KO mice. B) H&E staining of representative histological features of gastric mucosa from 2–4 month old mice; wild-type (n=9), TFF1 KO (n=14), DP KO (n=10), and TFF1 KO/DP KO (n=9). Lack of dysplastic gastric glands in wild-type, DP KO, and TFF1 KO/DP KO mice; dysplastic glands were observed in TFF1 KO mice (upper panel). Representative immune-histochemical staining of DARPP-32 and p-STAT3 in gastric tissues from wild-type, TFF1 KO, DP KO, and TFF1 KO/DP KO 2–4 month old mice (lower panel). C) Western blot analysis of IGFR-SRC pathway in 2–4 month old mice. D) Histological changes were examined in TFF1 KO and TFF1 KO/DP KO old mice. Wild-type mice (n=21), TFF1 KO (n=54), DP KO (n=11), and TFF1 KO/DP KO (n=37) mice were evaluated for chronic inflammation and dysplasia. Statistical significance in all panels was calculated by the 1-way ANOVA, followed by the Newman-Keuls test.

Article Snippet: Immunofluorescence was performed following cell fixation and permeabilization, using antibodies against DARPP-32 (1:400) (Santa Cruz, sc-398144), p-STAT3 (Cell Signaling, Y705, 9193s) or p-SRC (1:400) (Cell Signaling, Y416, 6943s).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Oncogene

Article Title: Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells

doi: 10.1038/s41388-019-0843-1

Figure Lengend Snippet: A) Representative images of immune-histochemical staining of DARPP-32 and p-STAT3 in tissue sections from human gastric mucosa with normal histology (NG, n=108) and adenocarcinoma (AdCa, n=108); original magnification ×20. B-C) The graphs summarize the immunohistochemical staining results on gastric tissue microarrays. D) Survival analysis of DARPP-32 and STAT3 mRNA expression in gastric cancer patients by the Kaplan-Meier survival curve, n=882, following analysis of public data online ( http://kmplot.com/analysis/index.php?p=service ) [25]. E) A diagram depicting the role of DARPP-32 in activation of STAT3 in gastric cancer cells. In summary, DARPP-32 interacts with IGF1R and promotes IGF1R and SRC phosphorylation, allowing sustained IL6-mediated phosphorylation and activation of STAT3. The translocation of p-STAT3 into the nucleus initiates transcriptional regulation of downstream target genes that regulate cell proliferation, transformation.

Article Snippet: Immunofluorescence was performed following cell fixation and permeabilization, using antibodies against DARPP-32 (1:400) (Santa Cruz, sc-398144), p-STAT3 (Cell Signaling, Y705, 9193s) or p-SRC (1:400) (Cell Signaling, Y416, 6943s).

Techniques: Staining, Immunohistochemical staining, Expressing, Activation Assay, Phospho-proteomics, Translocation Assay, Transformation Assay