Journal: Neurobiology of Stress

Article Title: Roles of corticotropin-releasing factor signaling in the lateral habenula in anxiety-like and alcohol drinking behaviors in male rats

doi: 10.1016/j.ynstr.2021.100395

Figure Lengend Snippet: Activation of CRF1R/CRF2R induces stronger changes in glutamate transmission in LHb neurons from EtOH-WD rats. Representative traces of mEPSCs in the absence (TTX) and presence of CRF1R agonist (Stressin I, A1) or CRF2R agonist (Urocortin, B1). Time course of Stressin I (A2) or Urocortin (B2) -induced changes in mEPSC frequency in Naïve (○) and EtOH-WD (△) rats. Summary of Stressin I (A3) and Urocortin (B3) induced changes in the frequency and amplitude of mEPSCs. @ p < 0.05, @@ p < 0.01, @@@ p < 0.001, Student's paired t -test for agonist vs. baseline. & p < 0.05 between Naïve and EtOH-WD rats.

Article Snippet: We purchased CRF, NBI27914, Stressin I, Astressin 2b,Urocortin, from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Transmission Assay

Journal: PLoS ONE

Article Title: Temporal Association of Herpes Simplex Virus ICP4 with Cellular Complexes Functioning at Multiple Steps in PolII Transcription

doi: 10.1371/journal.pone.0078242

Figure Lengend Snippet: Novel complexes copurifying with ICP4 (spectral counts).

Article Snippet: The antibodies used to probe the membranes were; N15, polyclonal rabbit serum, for ICP4 (1:500), Trap220/Med 1 (sc-8998-X), Med6 (sc-9434), Med 4 (NBP1-84977; Novus Biologicals), Med23 (550429; BD Bio), CDK8 (sc-1521), Med12 (NB100-2357; Novus Biologicals), Med 13 ((NB100-60642; Novus Biologicals), Med 26 (sc-166614), TAF1 (sc735), TBP (233-R Covance), p62 (sc292-X), p80/XPB (sc-20696-x), CPSF2 (sc-165983), CPSF7 (NB100-61600; Novus Biologicals), Brahma (sc-6450), CHD3 (ABE86; Milipore), RUVBL1 (06-1299; Milipore), RUVBL2 (06-1300; Milipore) at the manufacturers recommended dilutions.

Techniques:

Journal: PLoS ONE

Article Title: Temporal Association of Herpes Simplex Virus ICP4 with Cellular Complexes Functioning at Multiple Steps in PolII Transcription

doi: 10.1371/journal.pone.0078242

Figure Lengend Snippet: Western blot analysis of nuclear extracts (NE) or affinity purified (PD) samples collected at 6 hpi using either wtICP4 (KOS) or TAP-wtICP4 (TAP) expressing viruses. Antibodies directed against (A) p80 and p62 of TFIIH, (B) CPSF2 and CPSF7 of the cleavage and polyadenylation complex and (C) Brahma of Swi/Snf, CHD3 of the Nurd complex, and RuvBL1 and RuvBL2 of the Ino80 complex were used.

Article Snippet: The antibodies used to probe the membranes were; N15, polyclonal rabbit serum, for ICP4 (1:500), Trap220/Med 1 (sc-8998-X), Med6 (sc-9434), Med 4 (NBP1-84977; Novus Biologicals), Med23 (550429; BD Bio), CDK8 (sc-1521), Med12 (NB100-2357; Novus Biologicals), Med 13 ((NB100-60642; Novus Biologicals), Med 26 (sc-166614), TAF1 (sc735), TBP (233-R Covance), p62 (sc292-X), p80/XPB (sc-20696-x), CPSF2 (sc-165983), CPSF7 (NB100-61600; Novus Biologicals), Brahma (sc-6450), CHD3 (ABE86; Milipore), RUVBL1 (06-1299; Milipore), RUVBL2 (06-1300; Milipore) at the manufacturers recommended dilutions.

Techniques: Western Blot, Affinity Purification, Expressing

Journal: Cell Reports Medicine

Article Title: Unacylated-Ghrelin Impairs Hippocampal Neurogenesis and Memory in Mice and Is Altered in Parkinson’s Dementia in Humans

doi: 10.1016/j.xcrm.2020.100120

Figure Lengend Snippet:

Article Snippet: For IGF-1 and GH, platelet-rich plasma samples were analyzed using Human IGF-1 DuoSet ELISA (cat. No. DY291, R&D Systems), and Human GH DuoSet ELISA (Cat. No. DY1067, R&D Systems), using half-volume Nunclon Microwell 96-well plates.

Techniques: Clinical Proteomics, Recombinant, Saline, RNAscope, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software, Microscopy

Journal: Cancer immunology research

Article Title: Subversion of NK-cell and TNFα Immune Surveillance Drives Tumor Recurrence

doi: 10.1158/2326-6066.CIR-17-0175

Figure Lengend Snippet: TNFα-expanded MRD cells acquire the recurrence-competent phenotype. A, A total of 5 × 104 B16 cells or MRD B16 cells (expanded from a site of tumor injection for 72 hours in TNFα) were plated in triplicate. Twenty-four hours later, cDNA was analyzed by qRT-PCR for expression of YB-1 or TOPO-IIα. Relative quantities of mRNA were determined. *, P < 0.05; mean of the triplicate is shown. Representative of two separate experiments with two different B16 MRD recurrences. B–E, Skin explant from theB16tk cell injection site from mice treated with ganciclovir was plated with TNFα (B; 100 ng/mL), TNFα plus doxorubicin (C; 0.1 mg/mL), or cocultured with VEGF and 105 splenocytes and lymph node cells from C57BL/6 mice cleared of B16tk tumors after ganciclovir treatment without (D) or with doxorubicin (E). Seven days later, wells were inspected for actively growing tumor cells. Representative of three B16 MRD explants. F, A total of 103 B16 cells or MRD B16 cells (expanded from a site of tumor injection for 72 hours in TNFα) were plated in triplicate. Cells were infected with reovirus (MOI 1.0) in the presence or absence of IFNα (100 U) for 48 hours and titers of reovirus determined. Mean and SD of triplicates are shown, **, P < 0.001 (ANOVA).

Article Snippet: Cytokines and antibodies Cytokines and cytokine-neutralizing antibodies were added to cultures upon plating of the cells and used at the following concentrations in vitro : VEGF 165 (12 ng/mL; catalog no. CYF- 336; Prospec-Bio), TNFα (100 ng/mL; catalog no. 31501A; Pepro-tech), IL6 (100 pg/mL; catalog no. 216–16; PeproTech), anti-TNFα (0.4 μg/mL; catalog no. AF410NA; R&D Systems), universal IFNα (100 U; catalog no. 11200-2; R&D Systems), anti-IL6 (1 μg/mL; catalog no. MP5-20F3; BioLegend), LPS (25 ng/mL; catalog no. L4524; Sigma), and CpG (25 ng/mL; Mayo Clinic Oligonucleotide Core facility).

Techniques: Injection, Quantitative RT-PCR, Expressing, Infection

Journal:

Article Title: Insulin-like growth factor I receptor blockade enhances chemotherapy and radiation responses and inhibits tumour growth in human gastric cancer xenografts

doi: 10.1136/gut.2004.048926

Figure Lengend Snippet: Expression and roles of the insulin-like growth factor/insulin-like growth factor I receptor (IGF/IGF-Ir) axis in human gastric cancer cell lines. (A) Representative data of reverse transcription-polymerase chain reaction analyses. Both MKN45 and MKN74 cells expressed IGF-I message (396 bp) but neither MKN28 nor NUGC4 showed any expression. All cells expressed mRNAs of IGF-II (468 bp), IGF-Ir (755 bp), and insulin-like growth factor receptor 2 (IGF-2r) (430 bp). Controls were β-actin (540 bp) and GAPDH (300 bp). (B–E) After MKN45 cells were cultured to 60% confluence (1×106) in six well plates, medium was replaced with the indicated medium (complete medium (CM) or serum free medium (SFM)) for an additional 48 hours and then cell growth evaluated by trypan blue assay. Growth ratio was calculated compared with the cell number in complete medium with 10% fetal calf serum (1.76 (0.05)×106). Cell growth was suppressed by serum withdrawal (SFM, p<0.0001) but 100 ng/ml IGF-I ((B); p = 0.0230, SFM without IGF-I v SFM with IGF-I) and 100 ng/ml IGF-II ((C); p = 0.0227, SFM without IGF-II v SFM with IGF-II) partially restored growth. (D) Insulin-like growth factor binding protein 3 (IGFBP3) reduced cell growth in complete medium. (E) The growth suppressing effect of IGFBP3 was also seen in serum free medium dose dependently. (F–H) Both IGF-I (F) and IGF-II (100 ng/ml (G, H)) blocked induction of 5% ethanol (EtOH one hour) induced apoptosis in both MKN45 (F, G) and MKN74 (H). (F) p<0.0001, no stimulation v ethanol stimulation without IGF-I; p = 0.0003, ethanol without IGF-I v ethanol with 200 ng/ml IGF-I. (G) p = 0.0097, no stimulation v ethanol stimulation without IGF-II; p = 0.0179, ethanol without IGF-I v ethanol with IGF-I. (H) p = 0.0022, no stimulation v ethanol stimulation without IGF-II; p = 0.0033, ethanol without IGF-I v ethanol with IGF-I.

Article Snippet: Recombinant human IGF-I and IGF-II were purchased from R&D systems (Minneapolis, Minnesota, USA) and NH2 terminally truncated IGF-I (des(1–3) IGF-I) from GroPep (Adelaide, Australia).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Cell Culture, Binding Assay

Journal:

Article Title: Insulin-like growth factor I receptor blockade enhances chemotherapy and radiation responses and inhibits tumour growth in human gastric cancer xenografts

doi: 10.1136/gut.2004.048926

Figure Lengend Snippet: Downstream signals from insulin-like growth factor I receptor (IGF-Ir) in gastric cancer cells, MKN45 (A–F), NUGC4 (G, H), and MKN74 (I, J). (A) In MKN45, western blotting showed that 20 ng/ml insulin-like growth factor I (IGF-I) phosphorylated Akt-1, extracellular signal regulated kinase (ERK), and Bad. Both Akt and Bad phosphorylation was reduced by dominant negative forms (dns) of adenoviruses expressing IGF-Ir (Ad-IGF-Ir/dns, 30 multiplicity of infection (moi)). However, Ad-IGF-Ir/dns did not influence ERK-1/-2 phosphorylation to the same degree as Akt. pAkt, phosphorylated Akt-1; tAkt, total Akt-1; pERK, phosphorylated ERK-1/-2; tERK, total ERK-1/-2; pBad, phosphorylated Bad; tBad, total Bad. (B) Western blotting revealed that 10 ng/ml IGF-II phosphorylated Akt which was reduced by infection with adenoviruses expressing truncated IGF-Ir of 482 amino acids long (Ad-IGF-Ir/482st) (30 moi). (C) IGF-Ir/482st blocked p38 activity by p38 kinase assay. ATF2 is a substrate of p38 MAPK. (D) Insulin induces Akt phosphorylation which was not influenced by IGF-Ir/482st. (E) NH2 terminally truncated IGF-I (des(1-3)IGF-I 20 ng/ml) phosphorylated Akt to the same degree as IGF-I. Ad-IGF-Ir/482st reduced des(1-3)IGF-I inducing Akt phosphorylation. dIGF-I, des(1-3)IGF-I. (F) IGF-Ir/482st did not block Akt phosphorylation stimulated by high dose IGF-I, especially 200 ng/ml IGF-I. (G) In NUGC4, IGF-I stimulated phosphorylation of Akt and p38 mitogen activated protein kinase (MAPK) were blocked by both IGF-Ir/dns (30 moi) but phosphorylation of ERKs were not. p-p38, phosphorylated p38 MAPK; t-p38, total p38 MAPK. (H) IGF-Ir/dn blocked IGF-II induced Akt phosphorylation. (I, J) In MKN74, IGF induced Akt phosphorylation was blocked by 100 moi of Ad-IGF-Ir/482st, but phosphorylated ERK was not. LacZ, control (adenovirus expressing β-galactosidase).

Article Snippet: Recombinant human IGF-I and IGF-II were purchased from R&D systems (Minneapolis, Minnesota, USA) and NH2 terminally truncated IGF-I (des(1–3) IGF-I) from GroPep (Adelaide, Australia).

Techniques: Western Blot, Phospho-proteomics, Dominant Negative Mutation, Expressing, Infection, Activity Assay, Kinase Assay, Blocking Assay, Control