Journal: Carcinogenesis

Article Title: TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3

doi: 10.1093/carcin/bgw093

Figure Lengend Snippet: TGF-β-ALK5-Smad signaling induces CYR61 expression in pancreatic stellate cells. (A–C) Linear regression was performed using the microarray dataset GDS4103. n = 39 patient samples: (A) TGFB1, (B) SERPINE1 and (C) SMAD7. (D) Western blot of CYR61 (Abcam) in LTC-14 and imPSC cells that were serum starved in 1% FBS then treated with indicated doses of TGF-β1 for 16h. (E) Western blot of CYR61 (Abcam) in LTC-14 cells that were serum starved in 1% FBS then treated with 100 pM TGF-β for indicated times. (F) Quantitative RT-PCR for rCYR61 performed on LTC-14 cells treated with 100 pM TGF-β for 0, 3 or 6h. ANOVA ***P = 0.0002, Dunnett’s multiple comparison test, 0h versus 3h **P = 0.003, 0h versus 6h ***P = 0.0001. n = 3 replicates. (G) Western blot analyzing downstream TGF-β signaling. LTC-14 cells were serum starved in 1% FBS then treated with 100 pM TGF-β1 ligand for indicated times. (H) Western blot of CYR61 (Abcam) in LTC-14 cells pretreated with DMSO vehicle control or inhibitors against ALK5 (20 μM SB431542), p38 MAPK (10 µM SB203580) or PI3K (10 µM LY294002) for 30min then treated with 100 pM TGF-β1 for 16h. (I) Western blot for CYR61 (Abcam), Smad2 and Smad3 in LTC-14 cells stably expressing NTC or CRISPR constructs against both Smad2 and Smad3. All western blotting results are representative of three independent experiments.

Article Snippet: Antibodies against cleaved caspase 3 (9664), P-Smad2 (3101), Total Smad2 (3103), P-p38 MAPK (4511), Total p38 MAPK (9212), P-Akt (4058), Total Akt (4691) and Total Smad3 (9523) were all purchased from Cell Signaling Technology.

Techniques: Expressing, Microarray, Western Blot, Quantitative RT-PCR, Comparison, Control, Stable Transfection, CRISPR, Construct

Journal: The Journal of Clinical Investigation

Article Title: Type III TGF-? receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

doi: 10.1172/JCI69657

Figure Lengend Snippet: Cells were treated with doses of 10 ng/ml FGF2, 1 μM PD-173074, and 10 μM U0126. (A) Western blot for phosphorylated and total Erk. Differentiation markers after 72-hour TβRIII knockdown and rescue with nontargeted shRNA or shRNA against TβRIII, with or without 1 ng/ml FGF2 treatment (gray bars). Densitometry for pErk normalized to total Erk is shown as percent control. 5Y cells were transduced for 96 hours. Quantification of densitometry from 4 independent experiments is shown (normalized mean ± SEM). P < 0.001 for main effect receptor (2-way ANOVA); P < 0.0001 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (B) Western blots following 96 hours of TβRIII transduction and treatment. Densitometry for NF160 normalized to β-actin is shown as percent control. (C) Western blots following 96 hours of transduction with TβRIII or GFP control and dominant-negative FGFR1 (dnFGFR1) or IRES-GFP vector control. GFP fluorescence was used to verify construct expression. Densitometry for NF160 normalized to β-actin is shown as percent control.

Article Snippet: Human basic fibroblast growth factor (no. 8910) and the MEK 1/2 inhibitor U0126 (no. 9903) were purchased from Cell Signaling.

Techniques: Western Blot, Knockdown, shRNA, Control, Transduction, Dominant Negative Mutation, Plasmid Preparation, Fluorescence, Construct, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Type III TGF-? receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

doi: 10.1172/JCI69657

Figure Lengend Snippet: Cells were treated with doses of 10 ng/ml FGF2, 1 μM PD-173074, and 10 μM U0126. (A) Western blot for Id1 in stable SHEP cells serum-starved 24 hours prior to FGF2 treatment. Densitometry analysis for Id1 normalized to β-actin is shown as percent control. (B) Western blot for Id1 in SHEP cells transduced and treated with FGF2 for 72 hours. Densitometry for Id1 normalized to β-actin is shown as percent control. (C) Western blot for Id1 in 5Y cells transduced for 96 hours. Densitometry for Id1 normalized to β-actin is shown as percent control. (D) 5Y cells were transduced for 96 hours with TβRIII or GFP control and Id1 siRNA (siId1) or nontargeted control siRNA. Densitometry for NF160 normalized to β-actin is shown as percent control. (E) Microarray data set expression of ID1 in tumors with low (bottom 10%) and high (top 10%) TGFBR3 expression (median [horizontal bars] and interquartile range [boxes]). ****P < 0.0001 (Mann-Whitney). Linear regression analysis of ID1 expression, which was dependent on TGFBR3 expression, in the microarray data set.

Article Snippet: Human basic fibroblast growth factor (no. 8910) and the MEK 1/2 inhibitor U0126 (no. 9903) were purchased from Cell Signaling.

Techniques: Western Blot, Control, Microarray, Expressing, MANN-WHITNEY

Journal: The Journal of comparative neurology

Article Title: Expression profile analysis of vulnerable CA1 pyramidal neurons in young-middle aged Ts65Dn mice

doi: 10.1002/cne.23663

Figure Lengend Snippet: Antibody Characterization

Article Snippet: Negative controls include omission of primary and secondary antibodies as described previously ( Ginsberg 2010 ; Ginsberg et al. 2010a ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Name Structure Immunogen Antibody Information Concentration RmDo20 enzymatically dephosphorylated, purified rat NF-M Gift of VM-Y Lee; mouse monoclonal antibody 1:200 GRIA1 15 residue synthetic peptide (KMSHSSGMPLGATGL) corresponding to C-terminus of rat GluR1 Millipore/Upstate, 06-306; rabbit polyclonal antibody 1:1000 GRIA2/3 synthetic peptide corresponding to aa 864-883 of rat GluR2 Millipore, 06-307; rabbit polyclonal antibody; RRID:AB_2314574 1:1000 TrkB human TrkB aa 156-322 BD Transduction Laboratory, 610102; Mouse polyclonal antibody purified; RRID:AB_397508 1:500 TrkC peptide surrounding Gly50 of human TrkC Cell Signalling, 3376S, rabbit monoclonal antibody; RRID:AB_2155283 1:1000 NTF3 synthetic peptide from the C-terminal region of NTF3 conjugated to KLH Pierce/Thermo Scientific, PA514861; rabbit polyclonal antibody; RRID:AB_2154265 1:100 BDNF peptide mapping within an internal region of human BDNF Santa Cruz, SC-546; rabbit polyclonal antibody; RRID:AB_630940 1:1000 TUBB derived from 2-28-33 hybridoma Sigma; T-5293; mouse monoclonal antibody; RRID:AB_477580 1:1000 Open in a separate window Antibody Characterization

Techniques: Concentration Assay, Purification, Transduction, Derivative Assay

Journal: The Journal of comparative neurology

Article Title: Expression profile analysis of vulnerable CA1 pyramidal neurons in young-middle aged Ts65Dn mice

doi: 10.1002/cne.23663

Figure Lengend Snippet: (A) BDNF showed no difference in mRNA expression levels, however, the BDNF receptor TrkB (NTRK2) showed significant downregulation, in accordance with the microarray results. NTF3 showed a trend for downregulation and the NTF3 receptor TrkC (NTRK3) was significantly downregulated. ddCT was expressed as percent of 2N control for all qPCR results. Key, (* p < 0.05) and (t p < 0.1). Error bars indicate SEM.

Article Snippet: Negative controls include omission of primary and secondary antibodies as described previously ( Ginsberg 2010 ; Ginsberg et al. 2010a ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Name Structure Immunogen Antibody Information Concentration RmDo20 enzymatically dephosphorylated, purified rat NF-M Gift of VM-Y Lee; mouse monoclonal antibody 1:200 GRIA1 15 residue synthetic peptide (KMSHSSGMPLGATGL) corresponding to C-terminus of rat GluR1 Millipore/Upstate, 06-306; rabbit polyclonal antibody 1:1000 GRIA2/3 synthetic peptide corresponding to aa 864-883 of rat GluR2 Millipore, 06-307; rabbit polyclonal antibody; RRID:AB_2314574 1:1000 TrkB human TrkB aa 156-322 BD Transduction Laboratory, 610102; Mouse polyclonal antibody purified; RRID:AB_397508 1:500 TrkC peptide surrounding Gly50 of human TrkC Cell Signalling, 3376S, rabbit monoclonal antibody; RRID:AB_2155283 1:1000 NTF3 synthetic peptide from the C-terminal region of NTF3 conjugated to KLH Pierce/Thermo Scientific, PA514861; rabbit polyclonal antibody; RRID:AB_2154265 1:100 BDNF peptide mapping within an internal region of human BDNF Santa Cruz, SC-546; rabbit polyclonal antibody; RRID:AB_630940 1:1000 TUBB derived from 2-28-33 hybridoma Sigma; T-5293; mouse monoclonal antibody; RRID:AB_477580 1:1000 Open in a separate window Antibody Characterization

Techniques: Expressing, Microarray

Journal: The Journal of comparative neurology

Article Title: Expression profile analysis of vulnerable CA1 pyramidal neurons in young-middle aged Ts65Dn mice

doi: 10.1002/cne.23663

Figure Lengend Snippet: Immunoblot analysis using regional hippocampal dissections was performed to assess whether selected transcriptional alterations resulted in commensurate protein level changes in Ts65Dn mice compared to 2N littermates at 4-6 months of age. (A) Representative immunoblots for pro-BDNF, mature BDNF, NTF3, TrkB-FL, TrkB-T1, TrkC-FL, and TrkC-T1. β-tubulin (TUBB) expression was used as a loading control. (B) Normalized expression levels compared to TUBB expression show upregulation of pro-BDNF (p<0.02), a trend towards downregulation of TrkB-FL (p =0.09) and TrkC-T1 (p= 0.069), but no significant changes in NTF3, mature BDNF, TrkB-T1 and TrkC-FL expression. Key: 2N black, Ts65Dn grey.

Article Snippet: Negative controls include omission of primary and secondary antibodies as described previously ( Ginsberg 2010 ; Ginsberg et al. 2010a ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Name Structure Immunogen Antibody Information Concentration RmDo20 enzymatically dephosphorylated, purified rat NF-M Gift of VM-Y Lee; mouse monoclonal antibody 1:200 GRIA1 15 residue synthetic peptide (KMSHSSGMPLGATGL) corresponding to C-terminus of rat GluR1 Millipore/Upstate, 06-306; rabbit polyclonal antibody 1:1000 GRIA2/3 synthetic peptide corresponding to aa 864-883 of rat GluR2 Millipore, 06-307; rabbit polyclonal antibody; RRID:AB_2314574 1:1000 TrkB human TrkB aa 156-322 BD Transduction Laboratory, 610102; Mouse polyclonal antibody purified; RRID:AB_397508 1:500 TrkC peptide surrounding Gly50 of human TrkC Cell Signalling, 3376S, rabbit monoclonal antibody; RRID:AB_2155283 1:1000 NTF3 synthetic peptide from the C-terminal region of NTF3 conjugated to KLH Pierce/Thermo Scientific, PA514861; rabbit polyclonal antibody; RRID:AB_2154265 1:100 BDNF peptide mapping within an internal region of human BDNF Santa Cruz, SC-546; rabbit polyclonal antibody; RRID:AB_630940 1:1000 TUBB derived from 2-28-33 hybridoma Sigma; T-5293; mouse monoclonal antibody; RRID:AB_477580 1:1000 Open in a separate window Antibody Characterization

Techniques: Western Blot, Expressing

Journal: Molecular cell

Article Title: Histone variant and cell context determine H3K27M reprogramming of the enhancer landscape and oncogenic state

doi: 10.1016/j.molcel.2019.08.030

Figure Lengend Snippet: A) Differential H3K27Ac signal at p38 MAPK pathway gene MAPKAPK2. X-axis represents genomic location and y-axis represents RPM. Blue bars indicate differential enhancers and promoters activated in H3.1K27M over H3.3K27M Group A DIPG.

Article Snippet: Primary antibodies were incubated overnight in 1% BSA/TBST overnight at the following concentrations: PPIB (Abcam ab178397, 1:2000), total p38 MAPK (CST 9212S, 1:1000), phospho-p38 MAPK (CST 9211S, 1:1000).

Techniques:

Journal: Molecular cell

Article Title: Histone variant and cell context determine H3K27M reprogramming of the enhancer landscape and oncogenic state

doi: 10.1016/j.molcel.2019.08.030

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies were incubated overnight in 1% BSA/TBST overnight at the following concentrations: PPIB (Abcam ab178397, 1:2000), total p38 MAPK (CST 9212S, 1:1000), phospho-p38 MAPK (CST 9211S, 1:1000).

Techniques: Dot Blot, Immunofluorescence, Recombinant, Multiplex Assay, Activation Assay, Microarray, Expressing, Plasmid Preparation, Software