Journal: Nature Communications

Article Title: BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases

doi: 10.1038/ncomms16040

Figure Lengend Snippet: ( a ) RNA-sequencing in human monocyte-derived macrophages (hMDMs) shows mRNA copies of BCAT enzymes in normalized counts. n =14 healthy donor hMDMs were used for RNA-seq. ( b ) Chemical structure of ERG240 and leucine. ( c ) Enzymatic activity of recombinant human BCAT1 and BCAT2 in presence of ERG240. ( d ) Relative expression of IRG1 , HIF1A and IL1B measured by qRT-PCR in control, ERG240-treated (20 mM, 3 h), LPS treated (100 ng ml −1 , 3 h), and LPS+ERG240 treated (LPS, 100 ng ml −1 ; ERG240, 20 mM for 3 h) hMDMs. ns, non-significant. All expression values are normalized to those obtained for HPRT gene expression. n =3 healthy donor hMDMs were used in each group. ( e ) Western blot analysis of IRG1, IL-1β, HIF-1α and beta-actin (ACTB) in control (untreated), ERG240-treated (20 mM, 3 h), LPS treated (100 ng ml −1 , 3 h) and LPS+ERG240 treated (LPS, 100 ng ml −1 ; ERG240, 20 mM for 3 h) hMDMs. The experiment is representative of three independent experiments using n =3 healthy donor hMDMs each. ( f ) GC/MS results showing itaconic acid production in hMDMs in presence or absence of ERG240 (20 mM). LPS treatment was for 8 h (100 ng ml −1 ). GC/MS plot for each donor is shown separately where itaconate abundance denotes arbitrary units. Error bars are s.e.m. Significance was tested using two-tailed Student’s t -test or one-way ANOVA. * P <10 −3 following two-way ANOVA.

Article Snippet: Lipopolysaccharide was purchased from Sigma (LPS; Escherichia coli serotype 0111:B4; Cat No L4391) and the antibodies used in Western Blot analyses are as follows: IL-1β (Cell Signaling Technologies, #12703; D3U3E, dilution 1/1,000), IRG1 (Abcam, ab122624, rabbit polyclonal, dilution, 1/1,000), HIF-1α (NOVUS biological, NB100-449, rabbit polyclonal, dilution 1/2,000), ACTB (Santa Cruz, sc-47778, β-Actin (C4), mouse monoclonal, dilution 1/2,000), BCAT1 (anti Bcat1 raised in rabbit, produced in Susan Hutson’s laboratory , 8382, dilution 1/120,000), TUBB (Cell Signaling Technologies, #2128, rabbit monoclonal, dilution 1/30,000).

Techniques: RNA Sequencing, Derivative Assay, Activity Assay, Recombinant, Expressing, Quantitative RT-PCR, Control, Gene Expression, Western Blot, Gas Chromatography-Mass Spectrometry, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Reactive Astrogliosis Causes the Development of Spontaneous Seizures

doi: 10.1523/JNEUROSCI.1574-14.2015

Figure Lengend Snippet: Glutamate uptake is impaired in mice with chronic astrogliosis. a, Immunohistochemistry against the glutamate transporter Glt-1 and Western blot probing for Glt-1(b) and GLAST in the cortical gray matter of β1−/− (c) and control mice at different ages (4 and 6 weeks and >6 months). d, Glutamate uptake was assessed after puff application of 200 μm glutamate for 500 ms before and after the application of the glutamate transporter inhibitors TBOA and DHK. ACSF contained the inhibitors (inh.) TTX, AP-5, bicuculline, and CdCl2. TBOA/DHK-sensitive glutamate uptake was reduced in β1−/− astrocytes when compared with littermate controls. Whiskers in box-and-whisker plot show minimum to maximum values. The line in the middle of the box is plotted at the median, + indicates the mean. e, Protein expression levels of the astrocytic enzyme glutamine synthetase was unchanged in the cortical gray matter of β1−/− and control mice at 4 and 6 weeks of age, but reduced in both genotypes in mice older than 6 months. Error bars in bar graphs refer to SEM. Statistical significance with *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Primary antibodies used included mouse anti-GFAP (Millipore; 1:1000), rabbit anti-GFAP (Dako, #Z0334; 1:1000), chicken anti-GFAP (Millipore; 1:1000), rat anti-Cd11b (AbD; Serotec, #MCA711; 1:500), rabbit anti-Kir4.1 (Alomone, #APC-035; 1:500), rabbit anti-glutamine synthetase (Abcam, #ab16802; 1:500), rabbit anti-Glt-1 (anti-B12, rat Glt-1 residues 12–26; batch number “ab#360,” 0.2 mg/ml; 1:1000; Holmseth et al., 2005 ).

Techniques: Immunohistochemistry, Western Blot, Whisker Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2)

doi: 10.1074/jbc.RA119.008643

Figure Lengend Snippet: Enzymatic removal of VEGFR2 increases dimerization and activation. A, serum-starved semi-confluent VEGFR2/PAE control and PNGase F–treated (37 °C, 4 h) cells were stimulated with VEGF (100 ng/ml) for 0, 5, 10, and 30 min at 37 °C. Following treatment, cells were lysed and analyzed via Western blotting with phosphotyrosine 1054, total VEGFR2, and α-tubulin. At 0 min, p = 0.46 (not significant); at 5 min, p = 0.017; at 10 min, p = 4.1 × 10−4; and at 30 min, p = 0.038. B, in vitro kinase assay of VEGFR2 immunoprecipitated from VEGFR2/PAE cells. VEGFR2 was incubated at 37 °C, and PNGase F (500 units) was added for 1 h, followed by addition of ATP (0, 0.1, or 1 μm) for 15 min, followed by denaturation and Western blotting. At the 0 μm ATP, p = 0.17 (not significant); at 0.1 μm ATP, p = 0.022; and at 1 μm ATP, p = 0.025. C, VEGFR2/HUVEC(Tert) control and PNGase F–treated (37 °C, 4 h) cells stimulated with VEGF (100 ng/ml) for 0, 5, 10, and 30 min at 4 °C. At 0 min, p = 0.16 (not significant); at 5 min, p = 7.0 × 10−3; at 10 min, p = 0.041; and at 30 min, p = 0.020. For all experiments, Western blotting quantification of was performed using ImageJ (n = 3). All experiments were performed in triplicate. Controls are represented by circles, and PNGase F–treated samples are represented by triangles. Individual data points and standard deviations are shown. p values were calculated using a two-tailed t test. Rounded rectangles mark time points where statistically significant (p < 0.05) differences between the control and PNGase F–treated samples were observed.

Article Snippet: Glycerol-free PNGase F, Q5 high-fidelity DNA polymerase, T4 DNA ligase, NotI-HF, ClaI, and CutSmart buffer were obtained from New England Biolabs (Ipswich, MA).

Techniques: Activation Assay, Western Blot, In Vitro, Kinase Assay, Immunoprecipitation, Incubation, Two Tailed Test

Journal: Cell reports

Article Title: The HDAC-Associated Sin3B Protein Represses DREAM Complex Targets and Cooperates with APC/C to Promote Quiescence

doi: 10.1016/j.celrep.2018.11.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used for immunoblotting: mouse anti-Sin3B (Santa Cruz H-4 SC-13145), mouse anti-Sin3B (Santa Cruz H-5 SC-55516), rabbit anti-Sin3B (Santa Cruz AK-12), rabbit anti-Sin3A (Santa Cruz K-20), rabbit anti-KI67 (Millipore (Upstate) AB9260), mouse anti-a-tubulin (Sigma-Aldrich T9026), mouse anti-vinculin (Sigma Clone V9131), rabbit anti-vinculin (Cell Signaling 13901), rabbit anti-MYBL2 (Santa Cruz SC-724 N-19), mouse anti-p130 (BD Transduction Labs 610262), rabbit anti-p130 (Santa Cruz C-20 SC-317), mouse anti-CCND1 (Millipore/Calbiochem CC12), rabbit anti-CDKN1B (p27KIP1) (Cell Signaling 2552), rabbit anti-CCNA2 (Santa Cruz C-19 SC-596), mouse anti-CCNE1 (Santa Cruz HE12 SC247), rabbit anti-CCNF (Santa Cruz C-20 SC952), rabbit anti-CDKN1A (p21CIP1) (Santa Cruz C-19), mouse anti-GAPDH (Millipore MAB374), mouse anti-FLAG (M2 Sigma), mouse anti-HA (Sigma 12CA5), mouse anti-MYC tag (Cell Signaling 9B11), rabbit anti-HDAC1 (Cell Signaling D5C6VXP), rabbit anti-HDAC3 (Cell Sigaling #2632), and mouse anti-V5 (Invitrogen).

Techniques: Transduction, Purification, Recombinant, Protease Inhibitor, DC Protein Assay, Bicinchoninic Acid Protein Assay, Transfection, Knock-Out, Plasmid Preparation, Cloning, Software, CRISPR

Journal: PLoS ONE

Article Title: Agonist-Directed Desensitization of the β 2 -Adrenergic Receptor

doi: 10.1371/journal.pone.0019282

Figure Lengend Snippet: (a)–(d) Confocal images of cells after immunostained with anti-β 2 AR. The cells were first stimulated with 2 nM epinephrine for 1 min (a), 2 min (b), 5 min (c) and 30 min (d). After epinephrine removal, the cells were further incubated in medium at 37°C for 29 min (a), 28 min (b), 25 min (c) and 0 min (d), respectively, such that equal time post stimulation was achieved for all. The staining was performed using anti-β 2 AR antibody and Alexa Fluor 488 goat anti-rabbit antibody. The nuclei were stained with DAPI. Confocal microscopy images were acquired on Zeiss confocal laser scanning microscope (oil-immersion, 63× objective). The green color is from the β 2 AR, the blue from the nuclei. (e) The agonist exposure time and Ro31-8220 sensitivity of pERK stimulated by epinephrine in A431 cells. The cells were incubated with the vehicle (0 min) or 2 nM epinephrine for a specific time (1 min, 2 min, 30 min), followed by further incubation at 37°C for another time to ensure all reach 30 min post simulation. Ro31-8220 when used was presented throughout the incubation. Equal amounts of cell lysate were separated by SDS-PAGE and analyzed for pERK by Western blotting. Equivalent gel loading was confirmed by probing with antibodies against GAPDH.

Article Snippet: Rabbit polyclonal anti-β 2 AR (#sc-569), mouse monoclonal anti-pERK1/2 (#sc-81492) and rabbit polyclonal anti-GAPDH (#sc-25778) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Incubation, Staining, Confocal Microscopy, Laser-Scanning Microscopy, SDS Page, Western Blot