Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca 2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse / rat / human Substance P (rabbit polyclonal), Cy3 , Bioss USA , Cat # bs-0064R-Cy3.

Techniques: Functional Assay, Recombinant, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Inhibition, Fluorescence, SYBR Green Assay, Activation Assay, Bicinchoninic Acid Protein Assay, In Situ, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Software

Journal:

Article Title: BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma

doi: 10.1073/pnas.092123699

Figure Lengend Snippet: Mutually exclusive expression patterns of BORIS and CTCF correlate with epigenetic reprogramming in testis. (A and E) Immunostaining of thin (5-mm) sections of adult mouse testis with the affinity-purified anti-BORIS antibodies visualized by secondary alkaline phosphatase-conjugated rabbit anti-IgY antibodies. (B and F) Expression in adult mouse testis sections visualized using affinity-purified anti-N-CTCF antibodies and secondary alkaline phosphatase-conjugated antibodies. (C and D) Control staining obtained with normal chicken and rabbit serum, respectively. (G) BORIS-expressing cells in mouse tubule visualized by secondary FITC-labeled antibodies. (H) 4′,6-diamidino-2-phenylindole (DAPI)-inverted image of G. (I) Dual immunohistochemical staining by chicken ap-2-Ab anti-BORIS antibodies (Rhodamine fluorescence, red) and anti-5mC antibodies (FITC fluorescence, green). (J) Anti-5mC staining (FITC with DAPI counterstaining) at lower magnification. (K) Identification of the cell types with methylated DNA within the tubule, SG, Sertoli cells, and St. Note methylation free Sc. (L) An inverted DAPI image of K. (Magnification: A–D, 12-fold; E and F, 50-fold; G, H, K, and L, 106-fold; I and J, 44-fold.)

Article Snippet: For double-staining of chicken anti-BORIS and sheep anti-5mC Ab, a goat anti-chicken secondary Ab conjugated to biotin (Vector), with subsequent detection by avidin-Rhodamine (Vector), was used for BORIS detection, and rabbit anti-sheep Ab conjugated to FITC (Vector) for detection of 5mC.

Techniques: Expressing, Immunostaining, Affinity Purification, Staining, Labeling, Immunohistochemical staining, Fluorescence, Methylation

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1α expression precedes IL-1β after ischemic brain injury and is localised to areas of focal neuronal loss and penumbral tissues

doi: 10.1186/1742-2094-8-186

Figure Lengend Snippet: IL-1α is expressed by microglia localized to focal neuronal and BBB injury 24 h after MCAo . Images are coronal sections from brains of C57BL6/H and CX3CR1-GFP +/- mice 60 min MCAo and 24 h reperfusion. Widefield images show IL-1α-expressing (red), GFP positive (green) microglia in ipsilateral (Ai), not contralateral (ii) amygdala 24 h after MCAo in a CX3CR1-GFP +/- mouse. IL-1α immunohistochemistry with cresyl violet co-staining localises IL-1α expressing microglia to the peri-infarct zone in thalamus (Bi) and cortex (Bii) of a C57BL6/H mouse. Focal IgG staining (red) co-localized with IL-1α positive microglia (green) in the ipsilateral cortex of a C57BL6/H mouse (Ci). No IgG or IL-1α staining detected in the contralateral cortex (Cii). IL-1α positive microglia detected in larger areas of IgG staining in the ipsilateral (Ciii), but not contralateral (Civ) hemisphere. Co-localization of IL-1α positive microglia (red) with areas of neuronal loss (blue) in a C57BL6/H mouse (D). Occasional IL-1α positive microglia also found in areas where neurons were morphologically intact (D, inset). Confocal images (E) are maximum Z projections (Ei, iii) and confocal slices at the level of the nucleus (Eii, iv) of IL-1α expressing, GFP positive microglia in a CX3CR1-GFP +/- mouse. Cells with (Ei, ii), and without (Eiii, iv) nuclear IL-1α. Nuclear fluorescence intensities for IL-1α and GFP were quantified from confocal images, and the fold enrichment of IL-1α and GFP in microglial nuclei was calculated in comparison to whole cell fluorescence (F). All images are representative of n ≥ 3 mice. Quantification is of n = 4 CX3CR1-GFP +/- mouse brains, with each data point representing an individual cell, n ≥ 30 cells per brain.

Article Snippet: Staining of coronal brain sections from C57BL6/H mice (Figure ) and CX3CR1-GFP+/- mice (not shown) 24 h after MCAo for IL-1α and IgG (BA-2000, Vector Labs, biotinylated horse anti-mouse IgG, 2 μg/mL; S-32356, Invitrogen, Alexa 594 conjugated streptavidin, 5 μg/mL) revealed that IL-1α expressing cells co-localized to areas of focal BBB damage, mainly near the penumbral regions of the ipsilateral hemisphere (Figure ).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence

Journal: Science Advances

Article Title: Promoting the activation of T cells with glycopolymer-modified dendritic cells by enhancing cell interactions

doi: 10.1126/sciadv.abb6595

Figure Lengend Snippet: ( A ) Schematic of HTP transfection with a photo perforation transfection system (PTS) was used. ( B ) Representative images showing HTP transfection. Nuclei were stained by DAPI (blue), and the HA-tagged HTP was stained by FITC-avidin (green). Scale bar, 100 um. ( C ) Quantification of transfection efficiency compared with Lip2000. *** P < 0.001 compared with Lip2000. Data are means ± SEM ( n = 3). ( D ) Schematic of DC modification with glycopolymers. ( E ) Representative images showing green fluorescence on the DC cell surface. Nuclei were stained by DAPI (blue) and biotin-labeled poly-(MAG) (pMB) by FITC-avidin (green). ( F ) Representative images showing the modified DCs incubated in complete medium for specified times (1, 3, and 7 days). ( G ) Viability of engineered DC over the 7-day period. Data are means ± SEM ( n = 3). N.D., not determined.

Article Snippet: Primary antibody hemagglutinin (HA) and FITC-conjugated secondary antibody were from Wuhan Boster Biological Technology Ltd. (Wuhan, China).

Techniques: Transfection, Staining, Avidin-Biotin Assay, Modification, Fluorescence, Labeling, Incubation

Journal: Science Advances

Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

doi: 10.1126/sciadv.1600760

Figure Lengend Snippet: ( A ) BSP binds to the BRD acetyl-lysine cavity, allowing for further functionalization toward the front channel within the ZA loop (Ra vector annotated in orange) or the back of the pocket (Rb vector annotated in orange). The vectors are shown in the complex of BSP with BRD4(1). ( B ) Two variants of biotinylated BSP (BSP-a and BSP-b) were prepared to explore binding to human BRDs in cells by pull-down experiments. ( C ) Biotinylated BSP (BSP-a or BSP-b) immobilized on magnetic beads was used to pull down human CECR2 from Flp-In T-REx HEK293 cells stably expressing 3×FLAG CECR2. The protein captured from whole-cell lysate was identified using anti-FLAG. ( D ) Cell lysate from HEK293T cells was incubated with biotinylated BSP (BSP-a or BSP-b) immobilized on magnetic streptavidin beads in the presence or absence of 30 nmol of BSP for 2 hours at 4°C. After pull-down and tryptic digestion with trypsin, proteins were identified in a TripleTOF 5600 mass spectrometer. (Top) Normalized abundance of each BRD-containing protein in HEK293 cells (data from Proteomics DB; https://www.proteomicsdb.org/ ). (Bottom) Ratio of peptide to peptide abundance in the presence and absence of competing BSP, shown as a bar graph. BRD families are annotated with roman numerals. ( E ) FRAP evaluation of full-length GFP-tagged BRD4 dissociation from chromatin in U2OS cells. Nuclei of DMSO-treated (top) or BSP-treated (1 μM; bottom) cells. Target regions of photobleaching are indicated with a white circle. Scale bar, 10 μm. FL-BRD4, full-length BRD4; FL-BRD9, full-length BRD9. ( F ) Quantitative comparison of time to half-maximal fluorescence recovery for BRD4 FRAP studies using BSP (red bars) as a function of ligand concentration. ( G ) FRAP evaluation of full-length GFP-tagged BRD9 dissociation from chromatin in U2OS cells. Nuclei of DMSO-treated (top) or BSP-treated (1 μM; bottom) cells in the presence of 10 μM SAHA (added to increase the experimental window). Target regions of photobleaching are indicated with a white circle. Scale bars, 10 μm. ( H ) Quantitative comparison of time to half-maximal fluorescence recovery for BRD9 FRAP studies using BSP (red bars) as a function of ligand concentration. Data in (F) and (H) represent means ± SEM ( n = 30) and are annotated with P values obtained from a two-tailed t test (* P < 0.05 and *** P < 0.001).

Article Snippet: Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates.

Techniques: Plasmid Preparation, Binding Assay, Magnetic Beads, Stable Transfection, Expressing, Incubation, Mass Spectrometry, Comparison, Fluorescence, Concentration Assay, Two Tailed Test

Journal: Science Advances

Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

doi: 10.1126/sciadv.1600760

Figure Lengend Snippet: Δ T m shifts (°C) of biotinylated BSP (BSP-a and BSP-b) tested against a panel of BET and other diverse BRDs. Compounds (final concentration, 10 μM) were added to the proteins (final concentration, 2 μM); the temperature was increased from 25° to 96°C at a step of 3°C/min; excitation and emission filters for the SYPRO Orange dye were set to 465 and 590 nm; and experiments were performed in triplicate. Values are means ± SD.

Article Snippet: Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates.

Techniques: Concentration Assay

Journal: Science Advances

Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

doi: 10.1126/sciadv.1600760

Figure Lengend Snippet: Relative abundance of BRD-containing proteins in HEK293 cells (data taken from Proteomics DB; https://www.proteomicsdb.org/ ). Pull-down of human BRD-containing proteins with biotinylated BSP (BSP-a and BSP-b), followed by competitive elution with BSP and MS, resulted in enrichment of BSP-targeted BRDs.

Article Snippet: Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Intracellular Accumulation of Amyloid-β (Aβ) Protein Plays a Major Role in Aβ-Induced Alterations of Glutamatergic Synaptic Transmission and Plasticity

doi: 10.1523/JNEUROSCI.1201-14.2014

Figure Lengend Snippet: Twenty minute injection of Aβ42 does not significantly affect dendritic spine density of CA1 hippocampal neurons. A, B, Representative examples of CA1 neurons filled with biocytin and revealed with avidin conjugated to Alexa Fluor 488. Neuron shown in A was injected with vehicle, whereas neuron shown in B was injected with 200 nm Aβ42 for 20 min. Bottom boxes in A and B show high-magnification images of dendritic segments of cells in A and B, respectively. Scale bar, 3 μm. Alexa Fluor 488 fluorescence intensity (8-bit depth) was represented according to the color scale on the right (bottom = 0, top = 256). C, Bar graphs showing the mean number of dendritic spines per 100 μm. n.s.: p > 0.05.

Article Snippet: Subsequently, biocytin was revealed by incubating slices with Alexa Fluor 488-conjugated avidin (1:500 in blocking solution; Life Technologies) for 90 min at RT.

Techniques: Injection, Avidin-Biotin Assay, Fluorescence