Structured Review

Jackson Immuno neu n
Stereological analysis of dying cells. ( A ) Number of AIF+ cells exhibiting cytosolic or nuclear labeling in KO vs WT. AIF positivity was higher in KO animals than in WT animals for both nuclear AIF staining (indicating imminent or complete cellular death) or cytosolic AIF staining (indicating oxidative stress that may lead to death). ( B ) We found that an average of 87% (+/−23%) of the AIF+ cells are <t>Neu-N+</t> neurons. ( C ) Caspase positivity (indicating imminent or complete cellular death) is more prevalent in KO than in WT mice. ( D ) Caspase+ cells in KO mice lie nearer to vessels than Caspase- cells. Caspase+ cells in KO mice are nearer to vessels than Caspase+ cells in WT mice. See also SI Text 1, Tables S1 and S2 .
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Images

1) Product Images from "Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy"

Article Title: Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy

Journal: Scientific Reports

doi: 10.1038/srep43276

Stereological analysis of dying cells. ( A ) Number of AIF+ cells exhibiting cytosolic or nuclear labeling in KO vs WT. AIF positivity was higher in KO animals than in WT animals for both nuclear AIF staining (indicating imminent or complete cellular death) or cytosolic AIF staining (indicating oxidative stress that may lead to death). ( B ) We found that an average of 87% (+/−23%) of the AIF+ cells are Neu-N+ neurons. ( C ) Caspase positivity (indicating imminent or complete cellular death) is more prevalent in KO than in WT mice. ( D ) Caspase+ cells in KO mice lie nearer to vessels than Caspase- cells. Caspase+ cells in KO mice are nearer to vessels than Caspase+ cells in WT mice. See also SI Text 1, Tables S1 and S2 .
Figure Legend Snippet: Stereological analysis of dying cells. ( A ) Number of AIF+ cells exhibiting cytosolic or nuclear labeling in KO vs WT. AIF positivity was higher in KO animals than in WT animals for both nuclear AIF staining (indicating imminent or complete cellular death) or cytosolic AIF staining (indicating oxidative stress that may lead to death). ( B ) We found that an average of 87% (+/−23%) of the AIF+ cells are Neu-N+ neurons. ( C ) Caspase positivity (indicating imminent or complete cellular death) is more prevalent in KO than in WT mice. ( D ) Caspase+ cells in KO mice lie nearer to vessels than Caspase- cells. Caspase+ cells in KO mice are nearer to vessels than Caspase+ cells in WT mice. See also SI Text 1, Tables S1 and S2 .

Techniques Used: Labeling, Staining, Mouse Assay

2) Product Images from "Spatial Genetic Patterning of the Embryonic Neuroepithelium Generates GABAergic Interneuron Diversity in the Adult Cortex"

Article Title: Spatial Genetic Patterning of the Embryonic Neuroepithelium Generates GABAergic Interneuron Diversity in the Adult Cortex

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1629-07.2007

Activity of Cre recombinase in Nkx2.1–Cre/R26R–GFP transgenic embryos. A–D , Expression of GFP at four rostrocaudal levels in embryonic day 11.5 (E11.5) transgenic embryos. Arrow in B indicates the dorsal limit of GFP expression. Arrow in C shows expression in the AEP. The red box in B indicates the area shown in E–J . E–G , Comparison between the expression of the endogenous Nkx2.1 gene and GFP at E11.5. Nkx2.1 expression extends farther dorsally than GFP. H–J , Expression of Cre and GFP at E11.5. K–O , Expression of Lhx6 in E11.5 Nkx2.1–Cre/R26R–GFP transgenic embryos. Arrows indicate Lhx6 + GFP − cells in the dMGE ( L–O ). P–Q , GFP + cells in the cortex express Lhx6 (arrows). Arrowheads indicate blood vessels. Scale bars: A–D , 350 μm; E–J , 60 μm; K , 1250 μm; L–O , 25 μm; P–R , 15 μm.
Figure Legend Snippet: Activity of Cre recombinase in Nkx2.1–Cre/R26R–GFP transgenic embryos. A–D , Expression of GFP at four rostrocaudal levels in embryonic day 11.5 (E11.5) transgenic embryos. Arrow in B indicates the dorsal limit of GFP expression. Arrow in C shows expression in the AEP. The red box in B indicates the area shown in E–J . E–G , Comparison between the expression of the endogenous Nkx2.1 gene and GFP at E11.5. Nkx2.1 expression extends farther dorsally than GFP. H–J , Expression of Cre and GFP at E11.5. K–O , Expression of Lhx6 in E11.5 Nkx2.1–Cre/R26R–GFP transgenic embryos. Arrows indicate Lhx6 + GFP − cells in the dMGE ( L–O ). P–Q , GFP + cells in the cortex express Lhx6 (arrows). Arrowheads indicate blood vessels. Scale bars: A–D , 350 μm; E–J , 60 μm; K , 1250 μm; L–O , 25 μm; P–R , 15 μm.

Techniques Used: Activity Assay, Transgenic Assay, Expressing

Activity of Cre recombinase in Lhx6–Cre/R26R–YFP transgenic embryos and contribution of Lhx6 -expressing precursors to cortical interneuron populations in the adult mouse. A–D , Expression of GFP and Lhx6 in E16.6 transgenic embryos. E , F , The contribution of Lhx6 -expressing precursors to interneuron populations expressing CB, CR, PV, NPY, or SST in the motor and somatosensory cortex. The extent of colocalization between YFP and each of the five markers was quantified, and the data are presented as percentage of the total number of cells expressing each of the five markers. G , The number of Lhx6 -derived cells coexpressing YFP and one of the markers CB, CR, PV, NPY, or SST are presented as a percentage of the total number of YFP + cells. Scale bar: A , 100 μm; B–D , 20 μm.
Figure Legend Snippet: Activity of Cre recombinase in Lhx6–Cre/R26R–YFP transgenic embryos and contribution of Lhx6 -expressing precursors to cortical interneuron populations in the adult mouse. A–D , Expression of GFP and Lhx6 in E16.6 transgenic embryos. E , F , The contribution of Lhx6 -expressing precursors to interneuron populations expressing CB, CR, PV, NPY, or SST in the motor and somatosensory cortex. The extent of colocalization between YFP and each of the five markers was quantified, and the data are presented as percentage of the total number of cells expressing each of the five markers. G , The number of Lhx6 -derived cells coexpressing YFP and one of the markers CB, CR, PV, NPY, or SST are presented as a percentage of the total number of YFP + cells. Scale bar: A , 100 μm; B–D , 20 μm.

Techniques Used: Activity Assay, Transgenic Assay, Expressing, Derivative Assay

3) Product Images from "Eif-2a Protects Brainstem Motoneurons in a Murine Model of Sleep Apnea"

Article Title: Eif-2a Protects Brainstem Motoneurons in a Murine Model of Sleep Apnea

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.5232-07.2008

Ultrastructural localization of CHOP/GADD153 immunoreactivity in hypoglossal neurons. A–D , EM photomicrograph reveals CHOP/GADD153 silver-labeled particles (arrow) were rarely evident in ChAT-labeled somata ( A ; ChAT-S) or dendrites ( B ; ChAT-d)
Figure Legend Snippet: Ultrastructural localization of CHOP/GADD153 immunoreactivity in hypoglossal neurons. A–D , EM photomicrograph reveals CHOP/GADD153 silver-labeled particles (arrow) were rarely evident in ChAT-labeled somata ( A ; ChAT-S) or dendrites ( B ; ChAT-d)

Techniques Used: Labeling

Salubrinal increases phosphorylation of eIF-2a and prevents LTIH upregulation of CHOP/GADD153. A , Fluorescence photomicrograph of p-eIF-2a immunoreactivity (Alexa Fluor 594; red color) and ChAT immunoreactivity (Alexa Fluor 488; green color) in facial
Figure Legend Snippet: Salubrinal increases phosphorylation of eIF-2a and prevents LTIH upregulation of CHOP/GADD153. A , Fluorescence photomicrograph of p-eIF-2a immunoreactivity (Alexa Fluor 594; red color) and ChAT immunoreactivity (Alexa Fluor 488; green color) in facial

Techniques Used: Fluorescence

LTIH increase in CHOP/GADD153 immunoreactivity in both facial and hypoglossal but not in motor trigeminal neurons. The 50 μm vibratome sections, prepared for electron microscopic imaging and analysis with DAB-labeled ChAT and silver-enhanced gold-labeled
Figure Legend Snippet: LTIH increase in CHOP/GADD153 immunoreactivity in both facial and hypoglossal but not in motor trigeminal neurons. The 50 μm vibratome sections, prepared for electron microscopic imaging and analysis with DAB-labeled ChAT and silver-enhanced gold-labeled

Techniques Used: Imaging, Labeling

Proposed model of hypoxia/reoxygenation ER injury and protection. Motoneurons may be considered susceptible or resistant to injury from hypoxia/reoxygenation. Susceptible neurons have at baseline an unfolded protein response and activation of CHOP/GADD153,
Figure Legend Snippet: Proposed model of hypoxia/reoxygenation ER injury and protection. Motoneurons may be considered susceptible or resistant to injury from hypoxia/reoxygenation. Susceptible neurons have at baseline an unfolded protein response and activation of CHOP/GADD153,

Techniques Used: Activation Assay

4) Product Images from "Dermatopontin Interacts with Fibronectin, Promotes Fibronectin Fibril Formation, and Enhances Cell Adhesion *"

Article Title: Dermatopontin Interacts with Fibronectin, Promotes Fibronectin Fibril Formation, and Enhances Cell Adhesion *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.179762

Immunofluorescent images of Fn and DP in a fibrin clot. A fibrin clot was double stained with an anti-DP rabbit antibody ( A ) and anti-Fn mouse antibody ( B ), an anti-DP rabbit antibody ( D ), and an anti-fibrinogen mouse antibody ( E ). These antibodies were detected by a rhodamine-labeled anti-rabbit antibody and an FITC-labeled anti-mouse antibody, respectively. In the insets in A , B , D , and E , the primary antibodies were replaced with either preimmune serum or IgG, followed by incubation with the secondary antibodies. C and F , merged images of A and B and of D and E , respectively. Bars , 20 μm.
Figure Legend Snippet: Immunofluorescent images of Fn and DP in a fibrin clot. A fibrin clot was double stained with an anti-DP rabbit antibody ( A ) and anti-Fn mouse antibody ( B ), an anti-DP rabbit antibody ( D ), and an anti-fibrinogen mouse antibody ( E ). These antibodies were detected by a rhodamine-labeled anti-rabbit antibody and an FITC-labeled anti-mouse antibody, respectively. In the insets in A , B , D , and E , the primary antibodies were replaced with either preimmune serum or IgG, followed by incubation with the secondary antibodies. C and F , merged images of A and B and of D and E , respectively. Bars , 20 μm.

Techniques Used: Staining, Labeling, Incubation

5) Product Images from "Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth"

Article Title: Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200212046

mpc constitutively express 5 monocyte chemotactic factors. (A) RT-PCR analysis of mpc mRNA at day 14 of FKN, MDC, MCP-1, VEGF, and β2M (β2microglobulin). (B and C) ELISA for (B) MDC, MCP-1, and (C) VEGF of mpc supernatants. Each symbol represents one culture estimated in triplicate. (D–G) Immunolabeling of (D) FKN, (E) MDC, (F) MCP-1, (G) VEGF using FITC-conjugated secondary antibody. Blue, DAPI stain.
Figure Legend Snippet: mpc constitutively express 5 monocyte chemotactic factors. (A) RT-PCR analysis of mpc mRNA at day 14 of FKN, MDC, MCP-1, VEGF, and β2M (β2microglobulin). (B and C) ELISA for (B) MDC, MCP-1, and (C) VEGF of mpc supernatants. Each symbol represents one culture estimated in triplicate. (D–G) Immunolabeling of (D) FKN, (E) MDC, (F) MCP-1, (G) VEGF using FITC-conjugated secondary antibody. Blue, DAPI stain.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunolabeling, Staining

6) Product Images from "Agrin Binds to the Nerve-Muscle Basal Lamina via Laminin"

Article Title: Agrin Binds to the Nerve-Muscle Basal Lamina via Laminin

Journal: The Journal of Cell Biology

doi:

Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21 B8 . Simultaneously, 20 nM cN25 7 Fc were included. AChR aggregates were stained by rhodamine–α-bungarotoxin, and cN25 7 Fc bound to the myotubes was visualized with biotinylated goat anti–mouse IgG followed by fluorescein-conjugated streptavidin. cN25 7 Fc is concentrated in AChR clusters and is distributed along the edges of the myotubes ( arrowhead ). No staining is seen in the absence of this fragment (− cN25 7 Fc). Consistent with the idea that cN25 7 Fc binds to laminin, the distribution of myotube-bound cN25 7 Fc resembles the staining pattern obtained with anti-β/γ-specific antiserum 648 ( β / γ ). The β2 chain of laminin, also called s-laminin, is concentrated in AChR clusters. In light of the colocalization of cN25 7 Fc and AChR clusters, laminin-4 (α2, β2, γ1) is a binding partner of agrin in these clusters. Bar, 40 μm.
Figure Legend Snippet: Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21 B8 . Simultaneously, 20 nM cN25 7 Fc were included. AChR aggregates were stained by rhodamine–α-bungarotoxin, and cN25 7 Fc bound to the myotubes was visualized with biotinylated goat anti–mouse IgG followed by fluorescein-conjugated streptavidin. cN25 7 Fc is concentrated in AChR clusters and is distributed along the edges of the myotubes ( arrowhead ). No staining is seen in the absence of this fragment (− cN25 7 Fc). Consistent with the idea that cN25 7 Fc binds to laminin, the distribution of myotube-bound cN25 7 Fc resembles the staining pattern obtained with anti-β/γ-specific antiserum 648 ( β / γ ). The β2 chain of laminin, also called s-laminin, is concentrated in AChR clusters. In light of the colocalization of cN25 7 Fc and AChR clusters, laminin-4 (α2, β2, γ1) is a binding partner of agrin in these clusters. Bar, 40 μm.

Techniques Used: Binding Assay, Cell Culture, Staining

7) Product Images from "Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions"

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

Journal: Oncotarget

doi: 10.18632/oncotarget.23453

Effects of estradiol on translocation of estrogen receptor alpha (ERα) to mitochondria Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye (middle panel). Merged signals indicated that the ERα protein had been translocated into mitochondria (bottom panels). These fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p
Figure Legend Snippet: Effects of estradiol on translocation of estrogen receptor alpha (ERα) to mitochondria Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye (middle panel). Merged signals indicated that the ERα protein had been translocated into mitochondria (bottom panels). These fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p

Techniques Used: Translocation Assay, Staining

Effects of estradiol on translocation of estrogen receptor alpha (ERα) to nuclei Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Cellular nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals indicated that the ERα protein had been translocated into nuclei (bottom panel). These merged fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p
Figure Legend Snippet: Effects of estradiol on translocation of estrogen receptor alpha (ERα) to nuclei Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Cellular nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals indicated that the ERα protein had been translocated into nuclei (bottom panel). These merged fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p

Techniques Used: Translocation Assay, Staining

8) Product Images from "Evidence for a human leucocyte antigen-DM-induced structural change in human leucocyte antigen-DO?"

Article Title: Evidence for a human leucocyte antigen-DM-induced structural change in human leucocyte antigen-DO?

Journal: Immunology

doi: 10.1111/j.1365-2567.2008.02984.x

Transport-competent cDO molecules reveal the Mags.DO5 epitope independent of DM (a) Immunofluorescence microscopy analysis of cDO subcellular localization. HeLa DM-negative cells were stably transfected with either wild-type (wt) DO (a–d) or cDO chimera (e–h), permeabilized and incubated with Mag.DO5 and rabbit anti-calnexin followed by Alexa 488-labelled goat anti-mouse antibodies (a,e), biotinylated goat anti-rabbit antibody and Texas-red conjugated streptavidin (b,f). (c) and (g) show the merge of wt DO or cDO images with those obtained for calnexin. (d) and (h) show the cells in visible light. (b) Flow cytometry analysis of HeLa cells stably transfected with DO (left panel) or cDO (right panel). Cells were permeabilized and stained with Mags.DO5 or HKC5. (c) HEK293T cells were transfected with cDO and DM, lysed in Chaps or Triton X-100 (Triton) and DO was immunoprecipitated with the DOα-specific rabbit antiserum. Samples were analysed on immunoblots using the DMβ-specific rabbit antiserum. Control Raji cells were lysed in the same conditions and immunoprecipitation was performed for DO or for DR using XD5. (d) HEK293T cells were transiently transfected with DO or cDO in the absence or presence of DM. After 48 hr, cells were permeabilized and stained with Mags.DO5 and HKC5. Mean fluorescence values obtained for DM + and DM − cells were plotted as a ratio. These ratios are representative of at least two other experiments.
Figure Legend Snippet: Transport-competent cDO molecules reveal the Mags.DO5 epitope independent of DM (a) Immunofluorescence microscopy analysis of cDO subcellular localization. HeLa DM-negative cells were stably transfected with either wild-type (wt) DO (a–d) or cDO chimera (e–h), permeabilized and incubated with Mag.DO5 and rabbit anti-calnexin followed by Alexa 488-labelled goat anti-mouse antibodies (a,e), biotinylated goat anti-rabbit antibody and Texas-red conjugated streptavidin (b,f). (c) and (g) show the merge of wt DO or cDO images with those obtained for calnexin. (d) and (h) show the cells in visible light. (b) Flow cytometry analysis of HeLa cells stably transfected with DO (left panel) or cDO (right panel). Cells were permeabilized and stained with Mags.DO5 or HKC5. (c) HEK293T cells were transfected with cDO and DM, lysed in Chaps or Triton X-100 (Triton) and DO was immunoprecipitated with the DOα-specific rabbit antiserum. Samples were analysed on immunoblots using the DMβ-specific rabbit antiserum. Control Raji cells were lysed in the same conditions and immunoprecipitation was performed for DO or for DR using XD5. (d) HEK293T cells were transiently transfected with DO or cDO in the absence or presence of DM. After 48 hr, cells were permeabilized and stained with Mags.DO5 and HKC5. Mean fluorescence values obtained for DM + and DM − cells were plotted as a ratio. These ratios are representative of at least two other experiments.

Techniques Used: Immunofluorescence, Microscopy, Stable Transfection, Transfection, Incubation, Flow Cytometry, Cytometry, Staining, Immunoprecipitation, Western Blot, Fluorescence

9) Product Images from "Dynamics of Major Histocompatibility Complex Class II Compartments during B Cell Receptor-mediated Cell Activation"

Article Title: Dynamics of Major Histocompatibility Complex Class II Compartments during B Cell Receptor-mediated Cell Activation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011543

BcR stimulation downregulates cathepsin S activity in IIA1.6 cells. (A) Accumulation of the p10 invariant chain fragment in BcR-stimulated IIA1.6 cells. IIA1.6 cells were incubated for the times indicated with anti–mouse IgG antibodies, as described in Materials and Methods. The p10 fragment was detected with a rabbit antiserum specific for the cytoplasmic tail of the invariant chain. The same blot was incubated with an anti-IAα chain antiserum and then with an anti–β tubulin antibody (left). In the right panel, the p10 signal is normalized with the MHC class II signal obtained for the same lane and these p10 signals are expressed in arbitrary units. These results are representative of three independent experiments. (B) IIA1.6 cells were left unstimulated or were stimulated for various times with anti–mouse IgG antibodies and membranes were prepared to assess cathepsin S (top) β hexosaminidase (bottom right), and cathepsin B/L (bottom left) activities in fluorometric assays. Cathepsin S activity was tested at pH 7.0, because cathepsin S is functional at neutral pH, with (white bars) or without (back bars) LHVS, a cathepsin S inhibitor. Error bars correspond to the mean of three experiments. (C) Detection of cathepsin S with an iodinated peptide that binds to the active site of cathepsin S. Stimulated or unstimulated cells were incubated for 1 h with 125 I-Mu-Tyr-Ala-CH2F, with or without LHVS, and cell lysates were then subjected to electrophoresis in 12% polyacrylamide gels. Cathepsin S was detected as a 26-kD band. The labeling of this protein was inhibited by adding LHVS. LHVS did not inhibit the labeling of an higher molecular weight band in the gel at ∼32 kD, which was identified as cathepsin L. Rat basophilic leukemia cell line cells, which do not produce cathepsin S, were used as a negative control. In the bottom panel, each band was quantified with a PhosphorImager™. Cathepsin S labeling was normalized against labeling of the 32-kD band for each lane and cathepsin S labeling was quantified in arbitrary units.
Figure Legend Snippet: BcR stimulation downregulates cathepsin S activity in IIA1.6 cells. (A) Accumulation of the p10 invariant chain fragment in BcR-stimulated IIA1.6 cells. IIA1.6 cells were incubated for the times indicated with anti–mouse IgG antibodies, as described in Materials and Methods. The p10 fragment was detected with a rabbit antiserum specific for the cytoplasmic tail of the invariant chain. The same blot was incubated with an anti-IAα chain antiserum and then with an anti–β tubulin antibody (left). In the right panel, the p10 signal is normalized with the MHC class II signal obtained for the same lane and these p10 signals are expressed in arbitrary units. These results are representative of three independent experiments. (B) IIA1.6 cells were left unstimulated or were stimulated for various times with anti–mouse IgG antibodies and membranes were prepared to assess cathepsin S (top) β hexosaminidase (bottom right), and cathepsin B/L (bottom left) activities in fluorometric assays. Cathepsin S activity was tested at pH 7.0, because cathepsin S is functional at neutral pH, with (white bars) or without (back bars) LHVS, a cathepsin S inhibitor. Error bars correspond to the mean of three experiments. (C) Detection of cathepsin S with an iodinated peptide that binds to the active site of cathepsin S. Stimulated or unstimulated cells were incubated for 1 h with 125 I-Mu-Tyr-Ala-CH2F, with or without LHVS, and cell lysates were then subjected to electrophoresis in 12% polyacrylamide gels. Cathepsin S was detected as a 26-kD band. The labeling of this protein was inhibited by adding LHVS. LHVS did not inhibit the labeling of an higher molecular weight band in the gel at ∼32 kD, which was identified as cathepsin L. Rat basophilic leukemia cell line cells, which do not produce cathepsin S, were used as a negative control. In the bottom panel, each band was quantified with a PhosphorImager™. Cathepsin S labeling was normalized against labeling of the 32-kD band for each lane and cathepsin S labeling was quantified in arbitrary units.

Techniques Used: Activity Assay, Incubation, Functional Assay, Electrophoresis, Labeling, Molecular Weight, Negative Control

Redistribution of MHC class II–invariant chain complexes to lysosomes after BcR stimulation. (A) Percoll gradient fractionation of IIA1.6 cells and A20 cells expressing anti-DNP IgM. Cells were homogenized and the postnuclear supernatant (PNS) was fractionated on a 22% Percoll gradient. β hexosaminidase activity and alkaline phosphodiesterase activities were measured in each fraction and protein content was determined by Western blotting with anti-rab5, anti-rab7, anti-Lamp1, anti-H2-M, and anti-IAα chain antibodies. (B) Fractionation of MHC class II-containing compartments. Unstimulated and BcR-stimulated IIA1.6 cells (cells were incubated for 30 min at 37°C with anti–mouse IgG antibodies) were fractionated and β hexosaminidase and alkaline phosphodiesterase activities determined as in A. Pools of light (black bars) or dense (white bars) fractions were subjected to SDS-PAGE and MHC class II and invariant chain were quantified as described in Materials and Methods. The results shown are the means of three experiments. (C) Immunogold labeling of dense fractions containing MHC class II molecules after BCR stimulation. Membrane fractions were processed, immunogold-labeled, and contrast stained as described in Materials and Methods. (Top) The dense fractions are enriched in electron-dense compartments strongly labeled with an antibody directed against the cytoplasmic domain of IA. Internal membranes can be seen in the lumen of the electron-dense compartments. (Bottom) In the electron-dense fractions, small vesicles, 60–80 nm in diameter, are often observed. These vesicles, labeled with the M5114 antibody, probably correspond to the internal vesicles of multivesicular class II compartments. Their presence in these fractions is almost certainly due to the disruption of multivesicular bodies during fractionation. Scale bars, 100 nm. (D) DNP-coupled λ repressor was bound to A20 IgM anti-DNP cells at 4°C and the cells were incubated for 0 min, 30 min or 3 h at 37°C in complete medium. Cells were fractionated as below and we analyzed 24.4 T cell stimulation for the pools of fractions with β hexosaminidase or APDE activity, but similar amounts of proteins, by determining the IL- 2 content of the supernatant with a CTLL-2 assay.
Figure Legend Snippet: Redistribution of MHC class II–invariant chain complexes to lysosomes after BcR stimulation. (A) Percoll gradient fractionation of IIA1.6 cells and A20 cells expressing anti-DNP IgM. Cells were homogenized and the postnuclear supernatant (PNS) was fractionated on a 22% Percoll gradient. β hexosaminidase activity and alkaline phosphodiesterase activities were measured in each fraction and protein content was determined by Western blotting with anti-rab5, anti-rab7, anti-Lamp1, anti-H2-M, and anti-IAα chain antibodies. (B) Fractionation of MHC class II-containing compartments. Unstimulated and BcR-stimulated IIA1.6 cells (cells were incubated for 30 min at 37°C with anti–mouse IgG antibodies) were fractionated and β hexosaminidase and alkaline phosphodiesterase activities determined as in A. Pools of light (black bars) or dense (white bars) fractions were subjected to SDS-PAGE and MHC class II and invariant chain were quantified as described in Materials and Methods. The results shown are the means of three experiments. (C) Immunogold labeling of dense fractions containing MHC class II molecules after BCR stimulation. Membrane fractions were processed, immunogold-labeled, and contrast stained as described in Materials and Methods. (Top) The dense fractions are enriched in electron-dense compartments strongly labeled with an antibody directed against the cytoplasmic domain of IA. Internal membranes can be seen in the lumen of the electron-dense compartments. (Bottom) In the electron-dense fractions, small vesicles, 60–80 nm in diameter, are often observed. These vesicles, labeled with the M5114 antibody, probably correspond to the internal vesicles of multivesicular class II compartments. Their presence in these fractions is almost certainly due to the disruption of multivesicular bodies during fractionation. Scale bars, 100 nm. (D) DNP-coupled λ repressor was bound to A20 IgM anti-DNP cells at 4°C and the cells were incubated for 0 min, 30 min or 3 h at 37°C in complete medium. Cells were fractionated as below and we analyzed 24.4 T cell stimulation for the pools of fractions with β hexosaminidase or APDE activity, but similar amounts of proteins, by determining the IL- 2 content of the supernatant with a CTLL-2 assay.

Techniques Used: Fractionation, Expressing, Activity Assay, Western Blot, Incubation, SDS Page, Labeling, Staining, IA, Cell Stimulation

BcR stimulation modifies H2-M intracellular localization. (A) Intracellular accumulation of H2-M and MHC class II after BcR stimulation. IIA1.6 cells were not stimulated (top) or were stimulated (bottom) with anti–mouse IgG antibodies for 60 min. They were then fixed and processed for immunofluorescence staining. Cells were labeled with the biotinylated anti-IAd antibody MKD6 and a rabbit anti–H2-M antiserum. Bound antibody was detected with FITC-conjugated streptavidin and Texas Red–conjugated secondary antibodies, respectively. (B) Quantitative analysis of the distribution of H2-M during BCR stimulation was performed as in Fig. 4 . Results are presented as the percentage of gold particles in the various compartments.
Figure Legend Snippet: BcR stimulation modifies H2-M intracellular localization. (A) Intracellular accumulation of H2-M and MHC class II after BcR stimulation. IIA1.6 cells were not stimulated (top) or were stimulated (bottom) with anti–mouse IgG antibodies for 60 min. They were then fixed and processed for immunofluorescence staining. Cells were labeled with the biotinylated anti-IAd antibody MKD6 and a rabbit anti–H2-M antiserum. Bound antibody was detected with FITC-conjugated streptavidin and Texas Red–conjugated secondary antibodies, respectively. (B) Quantitative analysis of the distribution of H2-M during BCR stimulation was performed as in Fig. 4 . Results are presented as the percentage of gold particles in the various compartments.

Techniques Used: Immunofluorescence, Staining, Labeling

10) Product Images from "Chlamydia trachomatis Infection Inhibits Both Bax and Bak Activation Induced by Staurosporine "

Article Title: Chlamydia trachomatis Infection Inhibits Both Bax and Bak Activation Induced by Staurosporine

Journal: Infection and Immunity

doi: 10.1128/IAI.72.9.5470-5474.2004

Correlation of chlamydial antiapoptotic activity with chlamydial blockade of caspase 3 activation, mitochondrial cytochrome c release, and Bax activation. HeLa cells infected with C. trachomatis serovar L2 at an MOI of 0.5 for 40 h were induced to undergo apoptosis with staurosporine. The cultures were then fixed and processed for triple staining with antichlamydial antibodies plus Cy5 conjugates for chlamydial inclusions (blue), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling for fragmented DNA (green), various primary antibodies plus Cy3 conjugates for active caspase 3 (red; top row), mitochondrial cytochrome c release (red; middle row), or active Bax (red; bottom row). The images for the various colors were acquired individually with an Olympus confocal microscope and overlaid to make tricolor images. The bright granular red staining indicates mitochondrial localization of the labeled molecules. Note that in the cultures with both infection and apoptosis induction (last column), only uninfected cells and not infected cells were induced to undergo DNA fragmentation (panels d, h, and l), caspase 3 activation (panel d), mitochondrial cytochrome c release (panel h), and Bax activation (panel l).
Figure Legend Snippet: Correlation of chlamydial antiapoptotic activity with chlamydial blockade of caspase 3 activation, mitochondrial cytochrome c release, and Bax activation. HeLa cells infected with C. trachomatis serovar L2 at an MOI of 0.5 for 40 h were induced to undergo apoptosis with staurosporine. The cultures were then fixed and processed for triple staining with antichlamydial antibodies plus Cy5 conjugates for chlamydial inclusions (blue), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling for fragmented DNA (green), various primary antibodies plus Cy3 conjugates for active caspase 3 (red; top row), mitochondrial cytochrome c release (red; middle row), or active Bax (red; bottom row). The images for the various colors were acquired individually with an Olympus confocal microscope and overlaid to make tricolor images. The bright granular red staining indicates mitochondrial localization of the labeled molecules. Note that in the cultures with both infection and apoptosis induction (last column), only uninfected cells and not infected cells were induced to undergo DNA fragmentation (panels d, h, and l), caspase 3 activation (panel d), mitochondrial cytochrome c release (panel h), and Bax activation (panel l).

Techniques Used: Activity Assay, Activation Assay, Infection, Staining, End Labeling, Microscopy, Labeling

11) Product Images from "Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells"

Article Title: Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells

Journal: Virology Journal

doi: 10.1186/1743-422X-8-278

Transfected NSP4 travels to the cell surface in the absence of other viral proteins . A . BHK-21 cells were transfected with pcDNA3.2 NSP4 1-175 and surface biotinylated at 20 h post transfection at 4°C. Cells were lysed and surface proteins were precipitated with streptavidin agarose (left panel). Mock transfected cells were treated identically and served as controls (middle panel). Lanes 1 are the cell lysates from transfected or un-transfected cells and lanes 2 are the surface biotinylated, streptavidin pull downs of the transfected (left) or mock-transfected (center) cells. Lanes 3 show the transfected or mock-transfected lysates following biotinylation and streptavidin pull-down. RV-infected cell lysate (right panel) is shown as a NSP4 control. Note that substrate with femtogram sensitivity was used to detect the expression of transfected NSP4. B . BHK-21 cells were grown on glass cover slips, transfected with pcDNA3.2 NSP4 1-175 , surface biotinylated in the cold, and probed with affinity-purified anti-NSP4 150-175 and anti-rabbit IgG-CY2 (all panels) and either streptavidin-CY5 (top panels) or mouse anti- Na + /K + -ATPase and anti-mouse IgA-Texas Red (bottom panels,). Cells were visualized with a Stallion Digital Workstation. The colocalized images with surface molecules pseudo-colored red and NSP4 pixels pseudo-colored green are shown in panels 3. Yellow pixels indicate the areas of pixel overlap. On the far right is shown the antibody controls.
Figure Legend Snippet: Transfected NSP4 travels to the cell surface in the absence of other viral proteins . A . BHK-21 cells were transfected with pcDNA3.2 NSP4 1-175 and surface biotinylated at 20 h post transfection at 4°C. Cells were lysed and surface proteins were precipitated with streptavidin agarose (left panel). Mock transfected cells were treated identically and served as controls (middle panel). Lanes 1 are the cell lysates from transfected or un-transfected cells and lanes 2 are the surface biotinylated, streptavidin pull downs of the transfected (left) or mock-transfected (center) cells. Lanes 3 show the transfected or mock-transfected lysates following biotinylation and streptavidin pull-down. RV-infected cell lysate (right panel) is shown as a NSP4 control. Note that substrate with femtogram sensitivity was used to detect the expression of transfected NSP4. B . BHK-21 cells were grown on glass cover slips, transfected with pcDNA3.2 NSP4 1-175 , surface biotinylated in the cold, and probed with affinity-purified anti-NSP4 150-175 and anti-rabbit IgG-CY2 (all panels) and either streptavidin-CY5 (top panels) or mouse anti- Na + /K + -ATPase and anti-mouse IgA-Texas Red (bottom panels,). Cells were visualized with a Stallion Digital Workstation. The colocalized images with surface molecules pseudo-colored red and NSP4 pixels pseudo-colored green are shown in panels 3. Yellow pixels indicate the areas of pixel overlap. On the far right is shown the antibody controls.

Techniques Used: Transfection, Infection, Expressing, Affinity Purification

12) Product Images from "Modification of Proteins by Norepinephrine is Important for Vascular Contraction"

Article Title: Modification of Proteins by Norepinephrine is Important for Vascular Contraction

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2010.00131

Aorta and vena cava tissues can take up and attach NE-biotin to proteins. Freshly dissociated smooth muscle cells of the aorta (left) and vena cava (right) were incubated with either 12.7 μM NE-biotin or vehicle (not shown) for 30 min. Staining with a Rhodamine-Red-X conjugated streptavidin (red, bottom left) showed that NE-biotin entered the smooth muscle cells (indicated by α-actin staining, green, top right), and, in the vena cava, other vascular cells (red staining, bottom right of the vena cava). Nuclei are indicated by DAPI staining. Images are representative of tissues from four different rats.
Figure Legend Snippet: Aorta and vena cava tissues can take up and attach NE-biotin to proteins. Freshly dissociated smooth muscle cells of the aorta (left) and vena cava (right) were incubated with either 12.7 μM NE-biotin or vehicle (not shown) for 30 min. Staining with a Rhodamine-Red-X conjugated streptavidin (red, bottom left) showed that NE-biotin entered the smooth muscle cells (indicated by α-actin staining, green, top right), and, in the vena cava, other vascular cells (red staining, bottom right of the vena cava). Nuclei are indicated by DAPI staining. Images are representative of tissues from four different rats.

Techniques Used: Incubation, Staining

13) Product Images from "Frontal affinity chromatography analysis of constructs of DC‐SIGN, DC‐SIGNR and LSECtin extend evidence for affinity to agalactosylated N‐glycans"

Article Title: Frontal affinity chromatography analysis of constructs of DC‐SIGN, DC‐SIGNR and LSECtin extend evidence for affinity to agalactosylated N‐glycans

Journal: The Febs Journal

doi: 10.1111/j.1742-4658.2010.07792.x

Uptake of agalactosylated αAGP by CHO cells stably expressing DC‐SIGN, DC‐SIGNR and LSECtin. (A) CHO cells stably expressing DC‐SIGN, DC‐SIGNR and LSECtin were incubated with 10 μg·mL −1 of biotin‐labeled agalactosylated αAGP (blue line) and its intact form (green line) precomplexed with PE‐conjugated streptavidin on ice for 30 min, and allowed to internalize at 37 °C for 1 h in the presence or absence (orange line) of 2 m m CaCl 2 . Negative controls represent staining obtained using PE‐conjugated streptavidin (red line). Cells were analyzed by flow cytometry. Parental untransfected CHO cells were used as mock cells. (B) CHO cells expressing DC‐SIGN, DC‐SIGNR and LSECtin cells were internalized at 37 °C for the times shown with 10 μg·mL −1 of biotin‐labeled agalactosylated αAGP precomplexed with PE‐conjugated streptavidin.
Figure Legend Snippet: Uptake of agalactosylated αAGP by CHO cells stably expressing DC‐SIGN, DC‐SIGNR and LSECtin. (A) CHO cells stably expressing DC‐SIGN, DC‐SIGNR and LSECtin were incubated with 10 μg·mL −1 of biotin‐labeled agalactosylated αAGP (blue line) and its intact form (green line) precomplexed with PE‐conjugated streptavidin on ice for 30 min, and allowed to internalize at 37 °C for 1 h in the presence or absence (orange line) of 2 m m CaCl 2 . Negative controls represent staining obtained using PE‐conjugated streptavidin (red line). Cells were analyzed by flow cytometry. Parental untransfected CHO cells were used as mock cells. (B) CHO cells expressing DC‐SIGN, DC‐SIGNR and LSECtin cells were internalized at 37 °C for the times shown with 10 μg·mL −1 of biotin‐labeled agalactosylated αAGP precomplexed with PE‐conjugated streptavidin.

Techniques Used: Stable Transfection, Expressing, Incubation, Labeling, Staining, Flow Cytometry

14) Product Images from "The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis"

Article Title: The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky001

Purified Esco1 acetylates α-tubulin peptide in vitro . ( A ) Purification of Flag-tagged Esco1 proteins. Esco1 and enzymatically mutant Esco1-G768D were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco1-Flag and Esco1-G768D-Flag were identified by coomassie staining and western blotting using anti-Esco1 antibody. ( B ) The synthesized peptide of α-tubulin was identified by western blotting using Streptavidin-HRP. ( C ) In vitro acetylation assay with purified Esco1 proteins and synthesized peptide. Synthesized α-tubulin peptide was incubated with or without purified Esco1-Flag, Esco1-G768D-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-acetyl-α-tubulin (Lys 40) antibody for acetylation levels of α-tubulin and Streptavidin-HRP as a loading control.
Figure Legend Snippet: Purified Esco1 acetylates α-tubulin peptide in vitro . ( A ) Purification of Flag-tagged Esco1 proteins. Esco1 and enzymatically mutant Esco1-G768D were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco1-Flag and Esco1-G768D-Flag were identified by coomassie staining and western blotting using anti-Esco1 antibody. ( B ) The synthesized peptide of α-tubulin was identified by western blotting using Streptavidin-HRP. ( C ) In vitro acetylation assay with purified Esco1 proteins and synthesized peptide. Synthesized α-tubulin peptide was incubated with or without purified Esco1-Flag, Esco1-G768D-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-acetyl-α-tubulin (Lys 40) antibody for acetylation levels of α-tubulin and Streptavidin-HRP as a loading control.

Techniques Used: Purification, In Vitro, Mutagenesis, Staining, Western Blot, Synthesized, Acetylation Assay, Incubation

15) Product Images from "Bystander suppression of collagen-induced arthritis in mice fed ovalbumin"

Article Title: Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

Journal: Arthritis Research & Therapy

doi: 10.1186/ar1150

Effects on anti-bovine collagen type II (BCII) antibody responses in mice fed ovalbumin (OVA). Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with BCII or BCII mixed with OVA, emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. Numbers of IgG anti-BCII antibody-forming spleen cells (AFCs) (a) , and serum IgG (b) , IgG 1 (c) , and IgG 2a (d) anti-BCII antibody activity, 1 week after booster immunization. Each symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. * P
Figure Legend Snippet: Effects on anti-bovine collagen type II (BCII) antibody responses in mice fed ovalbumin (OVA). Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with BCII or BCII mixed with OVA, emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. Numbers of IgG anti-BCII antibody-forming spleen cells (AFCs) (a) , and serum IgG (b) , IgG 1 (c) , and IgG 2a (d) anti-BCII antibody activity, 1 week after booster immunization. Each symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. * P

Techniques Used: Mouse Assay, Activity Assay, MANN-WHITNEY

Effects on ovalbumin (OVA)-specific immune responses in mice fed OVA. Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with OVA or OVA mixed with bovine collagen type II (BCII) emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. (a) IgG anti-OVA antibody activity in serum 1 week after the booster immunization. Each circular symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. In vitro proliferation (b) and secretion of IFN-γ (c) , IL-4 (d) , and IL-10 (e) by splenocytes after OVA restimulation 1 week after the booster immunization. The proliferation results are presented as proliferation indexes (mean counts per minute [cpm] in triplicate cultures stimulated with OVA/mean cpm in triplicate control cultures). Bars represent mean ± standard error of the mean ( n = 7–9). * P
Figure Legend Snippet: Effects on ovalbumin (OVA)-specific immune responses in mice fed OVA. Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with OVA or OVA mixed with bovine collagen type II (BCII) emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. (a) IgG anti-OVA antibody activity in serum 1 week after the booster immunization. Each circular symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. In vitro proliferation (b) and secretion of IFN-γ (c) , IL-4 (d) , and IL-10 (e) by splenocytes after OVA restimulation 1 week after the booster immunization. The proliferation results are presented as proliferation indexes (mean counts per minute [cpm] in triplicate cultures stimulated with OVA/mean cpm in triplicate control cultures). Bars represent mean ± standard error of the mean ( n = 7–9). * P

Techniques Used: Mouse Assay, Activity Assay, MANN-WHITNEY, In Vitro

16) Product Images from "Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy"

Article Title: Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy

Journal: Scientific Reports

doi: 10.1038/srep43276

Stereological analysis of dying cells. ( A ) Number of AIF+ cells exhibiting cytosolic or nuclear labeling in KO vs WT. AIF positivity was higher in KO animals than in WT animals for both nuclear AIF staining (indicating imminent or complete cellular death) or cytosolic AIF staining (indicating oxidative stress that may lead to death). ( B ) We found that an average of 87% (+/−23%) of the AIF+ cells are Neu-N+ neurons. ( C ) Caspase positivity (indicating imminent or complete cellular death) is more prevalent in KO than in WT mice. ( D ) Caspase+ cells in KO mice lie nearer to vessels than Caspase- cells. Caspase+ cells in KO mice are nearer to vessels than Caspase+ cells in WT mice. See also SI Text 1, Tables S1 and S2 .
Figure Legend Snippet: Stereological analysis of dying cells. ( A ) Number of AIF+ cells exhibiting cytosolic or nuclear labeling in KO vs WT. AIF positivity was higher in KO animals than in WT animals for both nuclear AIF staining (indicating imminent or complete cellular death) or cytosolic AIF staining (indicating oxidative stress that may lead to death). ( B ) We found that an average of 87% (+/−23%) of the AIF+ cells are Neu-N+ neurons. ( C ) Caspase positivity (indicating imminent or complete cellular death) is more prevalent in KO than in WT mice. ( D ) Caspase+ cells in KO mice lie nearer to vessels than Caspase- cells. Caspase+ cells in KO mice are nearer to vessels than Caspase+ cells in WT mice. See also SI Text 1, Tables S1 and S2 .

Techniques Used: Labeling, Staining, Mouse Assay

17) Product Images from "Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin"

Article Title: Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.0209053

Intracellular CRAMP expression in blood and exudate PMN. (A) Western blot analysis of bPMN lysates using affinity-purified rabbit anti-CRAMP antibody. (B) Intracellular staining of pPMN with rabbit anti-CRAMP antibody followed by Cy3-conjugated donkey anti-rabbit antibody (i) and identical staining performed with antibody preincubated with excess of synthetic CRAMP peptide (ii). (C, Upper) PMN purified from blood (i), peritoneal exudate (ii), and TCF (iii) from C57BL/6 mice were stained intracellularly with biotinylated rabbit anti-CRAMP (black lines) or isotype control (gray lines) antibody, followed by RPE-conjugated Streptavidin and analyzed by flow cytometry. (iv) Purified TCF-PMN stained with the neutrophil marker NIMP-R14 (black line) or isotype control (gray line) antibody followed by FITC-conjugated goat anti-rat antibody. Graphs are representative of two to five independent experiments. (C, Lower) bPMN (i), pPMN (ii), and TCF-PMN (iii) were immunolabeled with rabbit anti-CRAMP or isotype control (not shown) antibody, followed by Cy3-conjugated donkey anti-rabbit antibody and examined by confocal microscopy. Isotype controls showed no detectable staining. Fluorescence micrographs (original magnification, ×100) are representatives of three independent experiments. FL 2, Fluorescence 2. (D) Total intracellular CRAMP in lysates of bPMN and pPMN of at least three C57BL/6 mice was determined by ELISA.
Figure Legend Snippet: Intracellular CRAMP expression in blood and exudate PMN. (A) Western blot analysis of bPMN lysates using affinity-purified rabbit anti-CRAMP antibody. (B) Intracellular staining of pPMN with rabbit anti-CRAMP antibody followed by Cy3-conjugated donkey anti-rabbit antibody (i) and identical staining performed with antibody preincubated with excess of synthetic CRAMP peptide (ii). (C, Upper) PMN purified from blood (i), peritoneal exudate (ii), and TCF (iii) from C57BL/6 mice were stained intracellularly with biotinylated rabbit anti-CRAMP (black lines) or isotype control (gray lines) antibody, followed by RPE-conjugated Streptavidin and analyzed by flow cytometry. (iv) Purified TCF-PMN stained with the neutrophil marker NIMP-R14 (black line) or isotype control (gray line) antibody followed by FITC-conjugated goat anti-rat antibody. Graphs are representative of two to five independent experiments. (C, Lower) bPMN (i), pPMN (ii), and TCF-PMN (iii) were immunolabeled with rabbit anti-CRAMP or isotype control (not shown) antibody, followed by Cy3-conjugated donkey anti-rabbit antibody and examined by confocal microscopy. Isotype controls showed no detectable staining. Fluorescence micrographs (original magnification, ×100) are representatives of three independent experiments. FL 2, Fluorescence 2. (D) Total intracellular CRAMP in lysates of bPMN and pPMN of at least three C57BL/6 mice was determined by ELISA.

Techniques Used: Expressing, Western Blot, Affinity Purification, Staining, Purification, Mouse Assay, Flow Cytometry, Cytometry, Marker, Immunolabeling, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay

18) Product Images from "Cbl-b Negatively Regulates B Cell Antigen Receptor Signaling in Mature B Cells through Ubiquitination of the Tyrosine Kinase Syk"

Article Title: Cbl-b Negatively Regulates B Cell Antigen Receptor Signaling in Mature B Cells through Ubiquitination of the Tyrosine Kinase Syk

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021686

Signaling active BCR caps are prolonged in Cbl-b −/− B cells. (a) Splenic B cells from Cbl-b −/− mice were incubated with a rat mAb specific for IgM at 4°C, washed and incubated with biotin-labeled goat antibodies specific for rat IgG and warmed to 37°C for increasing lengths of time. At the end of each time point the cells were fixed and incubated with Texas Red–conjugated streptavidin to detect the BCR. The cells were permeabilized and incubated with the phosphotyrosine-specific mAb PY20 detected using FITC-labeled F(ab′) 2 goat Abs specific for mouse IgG (b and c). Splenic B cells from Cbl-b −/− mice were incubated with RRX-conjugated Fab goat Abs specific for Igμ for 15 min at 25°C and the BCR was cross-linked by the addition of goat Abs specific for mouse IgM. The cells were incubated at 37°C for increasing lengths of time, permeabilized, and incubated with either rabbit phospho-Syk (Y519/520)-specific Abs (b) or with rabbit Syk-specific Abs (c) each detected using AlexaFluor 488-labeled goat Abs specific for rabbit IgG. In each case, the cells were examined by confocal laser scanning microscopy and shown are the merged images of the optimal single planes at a magnification of ×63. Shown are typical images of patch, Cap I and Cap II structures taken for phosphotyrosine images at 5 min for patch and Cap I structures and 30 min for Cap II structures and for Syk and phospho-Syk images at 0 min for patch, 2 min for Cap I, and 30 min for Cap II structures. The images were analyzed by the LSM 5 software program to quantify the colocalization of red and green fluorescence and the data plotted in histograms. (d) Purified splenic B cells from Cbl-b +/+ and Cbl-b −/− mice were treated as in panel a to cross-link the BCR and cells were incubated at 37°C for increasing lengths of time. At the end of each time point the cells were fixed with 3.7% paraformaldehyde, stained with Alexa 488-conjugated streptavidin, and examined by fluorescence microscopy. The mean percentage (±SD) of B cells exhibiting the patch, Cap I and Cap II structures with time after warming to 37°C is given. At least 500 cells at random were counted per time point.
Figure Legend Snippet: Signaling active BCR caps are prolonged in Cbl-b −/− B cells. (a) Splenic B cells from Cbl-b −/− mice were incubated with a rat mAb specific for IgM at 4°C, washed and incubated with biotin-labeled goat antibodies specific for rat IgG and warmed to 37°C for increasing lengths of time. At the end of each time point the cells were fixed and incubated with Texas Red–conjugated streptavidin to detect the BCR. The cells were permeabilized and incubated with the phosphotyrosine-specific mAb PY20 detected using FITC-labeled F(ab′) 2 goat Abs specific for mouse IgG (b and c). Splenic B cells from Cbl-b −/− mice were incubated with RRX-conjugated Fab goat Abs specific for Igμ for 15 min at 25°C and the BCR was cross-linked by the addition of goat Abs specific for mouse IgM. The cells were incubated at 37°C for increasing lengths of time, permeabilized, and incubated with either rabbit phospho-Syk (Y519/520)-specific Abs (b) or with rabbit Syk-specific Abs (c) each detected using AlexaFluor 488-labeled goat Abs specific for rabbit IgG. In each case, the cells were examined by confocal laser scanning microscopy and shown are the merged images of the optimal single planes at a magnification of ×63. Shown are typical images of patch, Cap I and Cap II structures taken for phosphotyrosine images at 5 min for patch and Cap I structures and 30 min for Cap II structures and for Syk and phospho-Syk images at 0 min for patch, 2 min for Cap I, and 30 min for Cap II structures. The images were analyzed by the LSM 5 software program to quantify the colocalization of red and green fluorescence and the data plotted in histograms. (d) Purified splenic B cells from Cbl-b +/+ and Cbl-b −/− mice were treated as in panel a to cross-link the BCR and cells were incubated at 37°C for increasing lengths of time. At the end of each time point the cells were fixed with 3.7% paraformaldehyde, stained with Alexa 488-conjugated streptavidin, and examined by fluorescence microscopy. The mean percentage (±SD) of B cells exhibiting the patch, Cap I and Cap II structures with time after warming to 37°C is given. At least 500 cells at random were counted per time point.

Techniques Used: Mouse Assay, Incubation, Labeling, Confocal Laser Scanning Microscopy, Software, Fluorescence, Purification, Staining, Microscopy

CDC42 activation and actin polymerization are enhanced in Cbl-b–deficient B cells. (A) Purified splenic B cells from Cbl-b +/+ and Cbl-b −/− mice were treated to cross-link the BCR as in Fig. 2, A–C , incubated at 37°C for the times indicated, fixed, permeabilized, and incubated with the WASP-GBD-GFP fusion protein followed by incubation with FITC-conjugated-rabbit antibodies specific for GFP and then FITC-conjugated goat antibodies specific for rabbit Ig. The cells were analyzed by flow cytometry and the results presented as the percent increase in mean fluorescence of activated B cells relative to the mean fluorescence of resting B cells. (B) B cells were treated as in panel A and permeabilized cells were stained with Alexa 488–conjugated phalloidin and analyzed by flow cytometry. Shown is the percent increase of the mean fluorescence of stimulated B cells relative to the mean fluorescence intensity of resting cells at each time point. (C) Splenic B cells from Cbl-b −/− mice were pretreated with the following inhibitors: piceatannol (100 μM for 1 h at 37°C) a Syk inhibitor(38); PP2 (100 μM for 1 h at 37°C) a Src-family kinase inhibitor (reference 37 ); and Cytochalasin D (10 μM for 1 h at 0°C; reference 36 ) or Latrunculin (10 μM for 30 min at 37°C; reference 36 ) inhibitors of the actin cytoskeleton. The pretreated cells were incubated with a rat mAb specific for IgM, washed and incubated for 2 or 10 min at 37°C with biotin-labeled F(ab′) 2 goat antibodies specific for rat IgG, fixed and stained with Alexa 488–labeled streptavidin to visualize the BCR. The cells were examined by fluorescence microscopy and the number of cells showing a patch, Cap I or Cap II morphology were scored.
Figure Legend Snippet: CDC42 activation and actin polymerization are enhanced in Cbl-b–deficient B cells. (A) Purified splenic B cells from Cbl-b +/+ and Cbl-b −/− mice were treated to cross-link the BCR as in Fig. 2, A–C , incubated at 37°C for the times indicated, fixed, permeabilized, and incubated with the WASP-GBD-GFP fusion protein followed by incubation with FITC-conjugated-rabbit antibodies specific for GFP and then FITC-conjugated goat antibodies specific for rabbit Ig. The cells were analyzed by flow cytometry and the results presented as the percent increase in mean fluorescence of activated B cells relative to the mean fluorescence of resting B cells. (B) B cells were treated as in panel A and permeabilized cells were stained with Alexa 488–conjugated phalloidin and analyzed by flow cytometry. Shown is the percent increase of the mean fluorescence of stimulated B cells relative to the mean fluorescence intensity of resting cells at each time point. (C) Splenic B cells from Cbl-b −/− mice were pretreated with the following inhibitors: piceatannol (100 μM for 1 h at 37°C) a Syk inhibitor(38); PP2 (100 μM for 1 h at 37°C) a Src-family kinase inhibitor (reference 37 ); and Cytochalasin D (10 μM for 1 h at 0°C; reference 36 ) or Latrunculin (10 μM for 30 min at 37°C; reference 36 ) inhibitors of the actin cytoskeleton. The pretreated cells were incubated with a rat mAb specific for IgM, washed and incubated for 2 or 10 min at 37°C with biotin-labeled F(ab′) 2 goat antibodies specific for rat IgG, fixed and stained with Alexa 488–labeled streptavidin to visualize the BCR. The cells were examined by fluorescence microscopy and the number of cells showing a patch, Cap I or Cap II morphology were scored.

Techniques Used: Activation Assay, Purification, Mouse Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Staining, Labeling, Microscopy

Phosphorylation of Igα and Syk and association of phospho-Syk with the BCR is prolonged in activated Cbl-b −/− B cells. (A) Purified splenic B cells from Cbl-b −/− and Cbl-b +/+ mice were treated to cross-link the BCR by incubation with a rat mAb specific for mouse IgM on ice for 30 min followed by incubation with F(ab′) 2 goat Abs specific for rat IgG at 37°C for the times indicated. At the end of each time point the cells were lysed, and the lysates subjected to immunoprecipitation using Igα-specific Abs. The Igα immunoprecipitates were analyzed by SDS-PAGE and immunoblotting probing for phosphotyrosine-containing proteins, stripped, and reprobed for either Igα (left panel) or Syk (right panel) using specific Abs. (B) B cells from Cbl-b +/+ and Cbl-b −/− mice were treated as in panel A to cross-link the BCR and at the end of each time point cells were lysed and Syk immunoprecipated from the lysates. The Syk immunoprecipitates were analyzed by SDS-PAGE and immunoblotting probing first with a phosphotyrosine-specific mAb, stripped, and reprobed for Syk. (C) Cbl-b +/+ and Cbl-b −/− B cells were treated in panel A to cross-link the BCR and incubated for 2 min at 37°C and lysed. Syk was immunoprecipitated from the lysates and the immunoprecipitates analyzed by SDS-PAGE and immunoblotting. The blots were first probed using phospho-Syk (Y519/520) specific Abs, stripped and reprobed with Syk-specific Abs. (D) B cells from Cbl-b +/− and Cbl-b −/− mice were imaged by laser scanning confocal microscopy to determine the colocalization of the BCR and phospho-Syk (Y519/520) as detailed in Materials and Methods. Briefly, B cells were incubated with RRX-conjugated Fab goat Abs specific for mouse Igμ, washed, allowed to settle onto coverslips, and activated by the addition of goat Abs specific for mouse IgM for 0 to 30 min at 37°C. The cells were fixed, permeabilized, and stained with rabbit Abs specific for phospho-Syk (Y519/520) detected using AlexaFluor 488-conjugated goat Abs specific for rabbit Ig. The cells were imaged by confocal laser scanning microscopy using a Zeiss Axiovert 200M LSM 510 META. A representative field is shown 2 min after BCR cross-linking (top) and the RRX and AlexaFluor 488 intensities of each pixel are plotted (bottom). (E) The average MFI of the AlexaFluor 488 colocalized with RRX for five fields of cells at each time point is given.
Figure Legend Snippet: Phosphorylation of Igα and Syk and association of phospho-Syk with the BCR is prolonged in activated Cbl-b −/− B cells. (A) Purified splenic B cells from Cbl-b −/− and Cbl-b +/+ mice were treated to cross-link the BCR by incubation with a rat mAb specific for mouse IgM on ice for 30 min followed by incubation with F(ab′) 2 goat Abs specific for rat IgG at 37°C for the times indicated. At the end of each time point the cells were lysed, and the lysates subjected to immunoprecipitation using Igα-specific Abs. The Igα immunoprecipitates were analyzed by SDS-PAGE and immunoblotting probing for phosphotyrosine-containing proteins, stripped, and reprobed for either Igα (left panel) or Syk (right panel) using specific Abs. (B) B cells from Cbl-b +/+ and Cbl-b −/− mice were treated as in panel A to cross-link the BCR and at the end of each time point cells were lysed and Syk immunoprecipated from the lysates. The Syk immunoprecipitates were analyzed by SDS-PAGE and immunoblotting probing first with a phosphotyrosine-specific mAb, stripped, and reprobed for Syk. (C) Cbl-b +/+ and Cbl-b −/− B cells were treated in panel A to cross-link the BCR and incubated for 2 min at 37°C and lysed. Syk was immunoprecipitated from the lysates and the immunoprecipitates analyzed by SDS-PAGE and immunoblotting. The blots were first probed using phospho-Syk (Y519/520) specific Abs, stripped and reprobed with Syk-specific Abs. (D) B cells from Cbl-b +/− and Cbl-b −/− mice were imaged by laser scanning confocal microscopy to determine the colocalization of the BCR and phospho-Syk (Y519/520) as detailed in Materials and Methods. Briefly, B cells were incubated with RRX-conjugated Fab goat Abs specific for mouse Igμ, washed, allowed to settle onto coverslips, and activated by the addition of goat Abs specific for mouse IgM for 0 to 30 min at 37°C. The cells were fixed, permeabilized, and stained with rabbit Abs specific for phospho-Syk (Y519/520) detected using AlexaFluor 488-conjugated goat Abs specific for rabbit Ig. The cells were imaged by confocal laser scanning microscopy using a Zeiss Axiovert 200M LSM 510 META. A representative field is shown 2 min after BCR cross-linking (top) and the RRX and AlexaFluor 488 intensities of each pixel are plotted (bottom). (E) The average MFI of the AlexaFluor 488 colocalized with RRX for five fields of cells at each time point is given.

Techniques Used: Purification, Mouse Assay, Incubation, Immunoprecipitation, SDS Page, Confocal Microscopy, Staining, Confocal Laser Scanning Microscopy

19) Product Images from "Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells"

Article Title: Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells

Journal: Virology Journal

doi: 10.1186/1743-422X-8-278

Transfected NSP4 travels to the cell surface in the absence of other viral proteins . A . BHK-21 cells were transfected with pcDNA3.2 NSP4 1-175 and surface biotinylated at 20 h post transfection at 4°C. Cells were lysed and surface proteins were precipitated with streptavidin agarose (left panel). Mock transfected cells were treated identically and served as controls (middle panel). Lanes 1 are the cell lysates from transfected or un-transfected cells and lanes 2 are the surface biotinylated, streptavidin pull downs of the transfected (left) or mock-transfected (center) cells. Lanes 3 show the transfected or mock-transfected lysates following biotinylation and streptavidin pull-down. RV-infected cell lysate (right panel) is shown as a NSP4 control. Note that substrate with femtogram sensitivity was used to detect the expression of transfected NSP4. B . BHK-21 cells were grown on glass cover slips, transfected with pcDNA3.2 NSP4 1-175 , surface biotinylated in the cold, and probed with affinity-purified anti-NSP4 150-175 and anti-rabbit IgG-CY2 (all panels) and either streptavidin-CY5 (top panels) or mouse anti- Na + /K + -ATPase and anti-mouse IgA-Texas Red (bottom panels,). Cells were visualized with a Stallion Digital Workstation. The colocalized images with surface molecules pseudo-colored red and NSP4 pixels pseudo-colored green are shown in panels 3. Yellow pixels indicate the areas of pixel overlap. On the far right is shown the antibody controls.
Figure Legend Snippet: Transfected NSP4 travels to the cell surface in the absence of other viral proteins . A . BHK-21 cells were transfected with pcDNA3.2 NSP4 1-175 and surface biotinylated at 20 h post transfection at 4°C. Cells were lysed and surface proteins were precipitated with streptavidin agarose (left panel). Mock transfected cells were treated identically and served as controls (middle panel). Lanes 1 are the cell lysates from transfected or un-transfected cells and lanes 2 are the surface biotinylated, streptavidin pull downs of the transfected (left) or mock-transfected (center) cells. Lanes 3 show the transfected or mock-transfected lysates following biotinylation and streptavidin pull-down. RV-infected cell lysate (right panel) is shown as a NSP4 control. Note that substrate with femtogram sensitivity was used to detect the expression of transfected NSP4. B . BHK-21 cells were grown on glass cover slips, transfected with pcDNA3.2 NSP4 1-175 , surface biotinylated in the cold, and probed with affinity-purified anti-NSP4 150-175 and anti-rabbit IgG-CY2 (all panels) and either streptavidin-CY5 (top panels) or mouse anti- Na + /K + -ATPase and anti-mouse IgA-Texas Red (bottom panels,). Cells were visualized with a Stallion Digital Workstation. The colocalized images with surface molecules pseudo-colored red and NSP4 pixels pseudo-colored green are shown in panels 3. Yellow pixels indicate the areas of pixel overlap. On the far right is shown the antibody controls.

Techniques Used: Transfection, Infection, Expressing, Affinity Purification

Confocal analyses of RV-infected HT29.F8 cells at 7 hpi . Panel A : Live HT29.F8 cells were surface biotinylated prior to fixation and permeabilization. Once the cells. were fixed and permeabilized, streptavidin-CY2 was utilized to label the biotinylated proteins on the exofacial PM (pseudo-colored red) and CY5-linked F(ab) 2 prepared from anti-NSP4 150-175 was added to label NSP4 (pseudo-colored green). The white, block arrow indicates an infected cell stained with anti-NSP4 150-175 -CY5-linked F(ab) 2 . The yellow arrows highlight the PM of the uninfected cells in which CY5-linked NSP4 150-175 F(ab) 2 is bound (green). Panel B is an enlargement of the white box in panel A and Panel C is the uninfected cell control that was labeled the same as the infected cells (panel A).
Figure Legend Snippet: Confocal analyses of RV-infected HT29.F8 cells at 7 hpi . Panel A : Live HT29.F8 cells were surface biotinylated prior to fixation and permeabilization. Once the cells. were fixed and permeabilized, streptavidin-CY2 was utilized to label the biotinylated proteins on the exofacial PM (pseudo-colored red) and CY5-linked F(ab) 2 prepared from anti-NSP4 150-175 was added to label NSP4 (pseudo-colored green). The white, block arrow indicates an infected cell stained with anti-NSP4 150-175 -CY5-linked F(ab) 2 . The yellow arrows highlight the PM of the uninfected cells in which CY5-linked NSP4 150-175 F(ab) 2 is bound (green). Panel B is an enlargement of the white box in panel A and Panel C is the uninfected cell control that was labeled the same as the infected cells (panel A).

Techniques Used: Infection, Blocking Assay, Staining, Labeling

20) Product Images from "UDP-Glucuronosyltransferase 1a Enzymes Are Present and Active in the Mouse Blastocyst"

Article Title: UDP-Glucuronosyltransferase 1a Enzymes Are Present and Active in the Mouse Blastocyst

Journal: Drug Metabolism and Disposition

doi: 10.1124/dmd.114.059766

Confocal immunofluorescence analysis of Ugt expression and localization in blastocysts. (A) Exemplary images of blastocysts stained with pan-specific antibodies against Ugt1a and Ugt2b, with DAPI staining to show the cell nuclei. Strong Ugt1a signal is
Figure Legend Snippet: Confocal immunofluorescence analysis of Ugt expression and localization in blastocysts. (A) Exemplary images of blastocysts stained with pan-specific antibodies against Ugt1a and Ugt2b, with DAPI staining to show the cell nuclei. Strong Ugt1a signal is

Techniques Used: Immunofluorescence, Expressing, Staining

21) Product Images from "mcl-1 Is an Immediate-Early Gene Activated by the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Signaling Pathway and Is One Component of the GM-CSF Viability Response"

Article Title: mcl-1 Is an Immediate-Early Gene Activated by the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Signaling Pathway and Is One Component of the GM-CSF Viability Response

Journal: Molecular and Cellular Biology

doi:

Mcl-1 induction requires the membrane-distal domain of the GM-CSF receptor β chain. (A) Schematic representation of human GM-CSF receptor mutants transfected into Ba/F3 cells. (B) Ba/F3 cells expressing various receptor mutants were depleted of cytokine for 20 h before they were stimulated with mIL-3 or hGM-CSF. At various times after stimulation (0.5 to 3 h), the cells were lysed and their total RNA was analyzed by Northern blotting with specific probes as indicated in the figure. (C) Equal numbers (2 × 10 6 ) of Ba/F3 cells expressing various GM-CSF receptor mutants were seeded for 24 h in 10 ml of medium containing no cytokine, mIL-3, or hGM-CSF, and then their genomic DNA was extracted and analyzed by agarose gel electrophoresis (2% agarose). (D) Cells treated as in panel C were stained with biotinylated annexin-V and Texas red-conjugated streptavidin as described in Materials and Methods. The positively stained (apoptotic) cells were quantified with Cytofluor 2350. The fluorescence units of the annexin-V-bound cells are plotted here to reflect the absolute numbers of apoptotic cells. □, S; , mIL-3; , hGM-CSF. (E) Mitogenic activity of Ba/F3 cells expressing various receptor mutants. Cells (10 4 ) cultured in medium containing no cytokine (S), mIL-3 or hGM-CSF were pulse-labeled with [ 3 H]thymidine for 20 min and lysed, and the incorporated counts were measured with a β-counter.
Figure Legend Snippet: Mcl-1 induction requires the membrane-distal domain of the GM-CSF receptor β chain. (A) Schematic representation of human GM-CSF receptor mutants transfected into Ba/F3 cells. (B) Ba/F3 cells expressing various receptor mutants were depleted of cytokine for 20 h before they were stimulated with mIL-3 or hGM-CSF. At various times after stimulation (0.5 to 3 h), the cells were lysed and their total RNA was analyzed by Northern blotting with specific probes as indicated in the figure. (C) Equal numbers (2 × 10 6 ) of Ba/F3 cells expressing various GM-CSF receptor mutants were seeded for 24 h in 10 ml of medium containing no cytokine, mIL-3, or hGM-CSF, and then their genomic DNA was extracted and analyzed by agarose gel electrophoresis (2% agarose). (D) Cells treated as in panel C were stained with biotinylated annexin-V and Texas red-conjugated streptavidin as described in Materials and Methods. The positively stained (apoptotic) cells were quantified with Cytofluor 2350. The fluorescence units of the annexin-V-bound cells are plotted here to reflect the absolute numbers of apoptotic cells. □, S; , mIL-3; , hGM-CSF. (E) Mitogenic activity of Ba/F3 cells expressing various receptor mutants. Cells (10 4 ) cultured in medium containing no cytokine (S), mIL-3 or hGM-CSF were pulse-labeled with [ 3 H]thymidine for 20 min and lysed, and the incorporated counts were measured with a β-counter.

Techniques Used: Transfection, Expressing, Northern Blot, Agarose Gel Electrophoresis, Staining, Fluorescence, Activity Assay, Cell Culture, Labeling

22) Product Images from "Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration"

Article Title: Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration

Journal: World Journal of Experimental Medicine

doi: 10.5493/wjem.v5.i2.140

Pax7 + cells in regenerating skeletal muscle implants. A, B: Examples of single (A and B) and pairs of (B) Pax7 + cells (arrows) attached or positioned in close proximity to myofibers in a fresh mouse implant at 14 d after implantation. Scale bar is 50
Figure Legend Snippet: Pax7 + cells in regenerating skeletal muscle implants. A, B: Examples of single (A and B) and pairs of (B) Pax7 + cells (arrows) attached or positioned in close proximity to myofibers in a fresh mouse implant at 14 d after implantation. Scale bar is 50

Techniques Used:

23) Product Images from "Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5 1"

Article Title: Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

Generation of anaphylatoxin-like molecules by the releasate of 22E7-stimulated skin mast cells. Supernatants were collected from two different batches of skin mast cells that had been activated by 22E7 (1 μ g/ml) for 30 min at 37°C and concentrated 2-fold with a Microcon YM-100 microconcentrator. Releasates were adjusted to pH 6.0 by adding 1/10 volume of 0.5 M MES buffer (pH 6.0). Approximately 8 μ l (60 ng of β -tryptase) of this releasate was incubated with 1 μ g of C3, C4, or C5, with or without B12 Fab (4 molar excess to β -tryptase) for 1 h at 37°C and then subjected to SDS-PAGE in 16% acrylamide gels. Western blotting was performed with rabbit anti-C3a, anti-C4a, or anti-C5a Ab, respectively. C5a′ is commercial C5a desArg .
Figure Legend Snippet: Generation of anaphylatoxin-like molecules by the releasate of 22E7-stimulated skin mast cells. Supernatants were collected from two different batches of skin mast cells that had been activated by 22E7 (1 μ g/ml) for 30 min at 37°C and concentrated 2-fold with a Microcon YM-100 microconcentrator. Releasates were adjusted to pH 6.0 by adding 1/10 volume of 0.5 M MES buffer (pH 6.0). Approximately 8 μ l (60 ng of β -tryptase) of this releasate was incubated with 1 μ g of C3, C4, or C5, with or without B12 Fab (4 molar excess to β -tryptase) for 1 h at 37°C and then subjected to SDS-PAGE in 16% acrylamide gels. Western blotting was performed with rabbit anti-C3a, anti-C4a, or anti-C5a Ab, respectively. C5a′ is commercial C5a desArg .

Techniques Used: Incubation, SDS Page, Western Blot

Effect of avidin on generation of C3a by polyanion-stabilized β -tryptase in the presence of B12 Fab. β -Tryptase (3.5 μ g/ml) stabilized with limiting amounts of polyanions was incubated with a 4 molar excess of B12 Fab and 33 μ g/ml of C3 at 37°C for 20 min at pH 6.0 in PBS with and without 12.5 μ g/ml of avidin. After SDS-PAGE in a 16% acryl-amide gel and Western blotting, C3a was probed with rabbit anti-C3a Ab. Contents were of HMW heparin (HMW-h), LMW heparin (LMW-h), commercial C3 control (C3), commercial C3a control (C3a), and degraded C3a (*).
Figure Legend Snippet: Effect of avidin on generation of C3a by polyanion-stabilized β -tryptase in the presence of B12 Fab. β -Tryptase (3.5 μ g/ml) stabilized with limiting amounts of polyanions was incubated with a 4 molar excess of B12 Fab and 33 μ g/ml of C3 at 37°C for 20 min at pH 6.0 in PBS with and without 12.5 μ g/ml of avidin. After SDS-PAGE in a 16% acryl-amide gel and Western blotting, C3a was probed with rabbit anti-C3a Ab. Contents were of HMW heparin (HMW-h), LMW heparin (LMW-h), commercial C3 control (C3), commercial C3a control (C3a), and degraded C3a (*).

Techniques Used: Avidin-Biotin Assay, Incubation, SDS Page, Western Blot

Time course of C3a-like molecule generation by β -tryptase stabilized with DS5K ( A ) and HMW heparin ( B ). β -Tryptase (3.5 μ g/ml) was stabilized with 10 μ g/ml DS5K or 0.8 μ g/ml HMW heparin and incubated with a 4 molar excess of B12 Fab and then 33 μ g/ml C3 in pH 6.0 PBS at 37°C at indicated time. After SDS-PAGE in 16% acrylamide gels, Western blotting was performed with rabbit anti-C3a Ab. Time points include 0, 1, 2, 3, 4, 5, 10, 20, 30, 60, and 120 min ( lanes 1–11 ) with a commercial C3a control (C3a) ( lane 12 ).
Figure Legend Snippet: Time course of C3a-like molecule generation by β -tryptase stabilized with DS5K ( A ) and HMW heparin ( B ). β -Tryptase (3.5 μ g/ml) was stabilized with 10 μ g/ml DS5K or 0.8 μ g/ml HMW heparin and incubated with a 4 molar excess of B12 Fab and then 33 μ g/ml C3 in pH 6.0 PBS at 37°C at indicated time. After SDS-PAGE in 16% acrylamide gels, Western blotting was performed with rabbit anti-C3a Ab. Time points include 0, 1, 2, 3, 4, 5, 10, 20, 30, 60, and 120 min ( lanes 1–11 ) with a commercial C3a control (C3a) ( lane 12 ).

Techniques Used: Incubation, SDS Page, Western Blot

Mass spectrometry analysis of anaphylatoxin-like molecules. C5a-like, C4a-like, and C3a-like molecules, respectively, were generated by β -tryptase with B12 Fab in PBS (pH 6.0) that had been stabilized with DS500K, LMW heparin, and DS5K and then incubated with parent complement proteins for 40, 60, and 20 min. C5a-like molecules were further treated with N -glycosidase F for 18 h. These anaphylatoxin-like molecules were concentrated by ZipTip C18 reversed-phase chromatography and subjected to mass spectrometry using a MALDI system.
Figure Legend Snippet: Mass spectrometry analysis of anaphylatoxin-like molecules. C5a-like, C4a-like, and C3a-like molecules, respectively, were generated by β -tryptase with B12 Fab in PBS (pH 6.0) that had been stabilized with DS500K, LMW heparin, and DS5K and then incubated with parent complement proteins for 40, 60, and 20 min. C5a-like molecules were further treated with N -glycosidase F for 18 h. These anaphylatoxin-like molecules were concentrated by ZipTip C18 reversed-phase chromatography and subjected to mass spectrometry using a MALDI system.

Techniques Used: Mass Spectrometry, Generated, Incubation, Reversed-phase Chromatography

C3 cleavage by β -tryptase stabilized with limiting amounts of different polyanions in the presence or absence of B12 Fab. A , C3a Western blotting. β -Tryptase (3.5 μ g/ml) was mixed with equal volumes of HMW heparin (0.8 μ g/ml), LMW heparin (7 μ g/ml), DS500K (0.7 μ g/ml), or DS5K (10 μ g/ml) and incubated for 10 min at room temperature, then for another 15 min with a 4 molar excess of B12 Fab or MOPC Fab, as indicated, and finally for 15 min at 37°C with 33 μ g/ml human C3 in PBS at pH 6.0. Samples were then subjected to SDS-PAGE in a 16% acrylamide gel followed by Western blotting using rabbit anti-C3a Ab as described. C3 ( lane 9 ) and C3a ( lane 10 ) controls are shown. B , Cleavage of C3 α but not C3 β by β -tryptase. Protein bands were detected by staining with Coomassie brilliant blue after SDS-PAGE in a 12% acrylamide gel. Lane contents were as described.
Figure Legend Snippet: C3 cleavage by β -tryptase stabilized with limiting amounts of different polyanions in the presence or absence of B12 Fab. A , C3a Western blotting. β -Tryptase (3.5 μ g/ml) was mixed with equal volumes of HMW heparin (0.8 μ g/ml), LMW heparin (7 μ g/ml), DS500K (0.7 μ g/ml), or DS5K (10 μ g/ml) and incubated for 10 min at room temperature, then for another 15 min with a 4 molar excess of B12 Fab or MOPC Fab, as indicated, and finally for 15 min at 37°C with 33 μ g/ml human C3 in PBS at pH 6.0. Samples were then subjected to SDS-PAGE in a 16% acrylamide gel followed by Western blotting using rabbit anti-C3a Ab as described. C3 ( lane 9 ) and C3a ( lane 10 ) controls are shown. B , Cleavage of C3 α but not C3 β by β -tryptase. Protein bands were detected by staining with Coomassie brilliant blue after SDS-PAGE in a 12% acrylamide gel. Lane contents were as described.

Techniques Used: Western Blot, Incubation, SDS Page, Acrylamide Gel Assay, Staining

C3 conversion and generation of C3a in plasma by β -tryptase. A , β -Tryptase-catalyzed conversion of C3 in plasma to C3a in the presence of B12 Fab at pH 6.0 in PBS. Samples were subjected to SDS-PAGE in 8% acrylamide gels, and Western blotted using goat anti-C3 Ab. Human plasma (1 μ l) was incubated with 320, 150, and 80 ng of DS500K-stabilized β -tryptase for 30 min. B , Time-dependent generation of C3a from plasma C3 by DS500K-stabilized β -tryptase (320 ng) detected by SDS-PAGE in 16% acrylamide gels followed by Western blotting using rabbit anti-C3a Ab. C3a is commercial C3a control.
Figure Legend Snippet: C3 conversion and generation of C3a in plasma by β -tryptase. A , β -Tryptase-catalyzed conversion of C3 in plasma to C3a in the presence of B12 Fab at pH 6.0 in PBS. Samples were subjected to SDS-PAGE in 8% acrylamide gels, and Western blotted using goat anti-C3 Ab. Human plasma (1 μ l) was incubated with 320, 150, and 80 ng of DS500K-stabilized β -tryptase for 30 min. B , Time-dependent generation of C3a from plasma C3 by DS500K-stabilized β -tryptase (320 ng) detected by SDS-PAGE in 16% acrylamide gels followed by Western blotting using rabbit anti-C3a Ab. C3a is commercial C3a control.

Techniques Used: SDS Page, Western Blot, Incubation

24) Product Images from "Rewiring of Afferent Fibers in the Somatosensory Thalamus of Mice Caused by Peripheral Sensory Nerve Transection"

Article Title: Rewiring of Afferent Fibers in the Somatosensory Thalamus of Mice Caused by Peripheral Sensory Nerve Transection

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.5008-11.2012

The IONC operation causes small but significant increase in GluA2 expression in the contralateral VPM. A , A photomontage at low magnification showing a mild increase in GluA2 immunoreactivity in the contralateral VPM. B , C , Images at high magnification showing double immunofluorescence for GluA2 (red) and VGluT2 (green) in the ipsilateral ( B 1 ) and contralateral ( C 1 ) VPM and single immunofluorescence for GluA2 ( B 2 , C 2 ), respectively. Note that fluorescence intensity of VGluT2-associated GluA2 immunolabeling (indicated by arrows) is increased in the contralateral VPM. Hi (DG), Hippocampus (dentate gyrus); Rt, reticular nucleus of thalamus; Str, striatum; contra, contralateral; ipsi, ipsilateral. Scale bars: A , 1 mm; (in C 2 ) B and C , 5 μm.
Figure Legend Snippet: The IONC operation causes small but significant increase in GluA2 expression in the contralateral VPM. A , A photomontage at low magnification showing a mild increase in GluA2 immunoreactivity in the contralateral VPM. B , C , Images at high magnification showing double immunofluorescence for GluA2 (red) and VGluT2 (green) in the ipsilateral ( B 1 ) and contralateral ( C 1 ) VPM and single immunofluorescence for GluA2 ( B 2 , C 2 ), respectively. Note that fluorescence intensity of VGluT2-associated GluA2 immunolabeling (indicated by arrows) is increased in the contralateral VPM. Hi (DG), Hippocampus (dentate gyrus); Rt, reticular nucleus of thalamus; Str, striatum; contra, contralateral; ipsi, ipsilateral. Scale bars: A , 1 mm; (in C 2 ) B and C , 5 μm.

Techniques Used: Expressing, Immunofluorescence, Fluorescence, Immunolabeling

25) Product Images from "Heat shock protein 70 expression, keratin phosphorylation and Mallory body formation in hepatocytes from griseofulvin-intoxicated mice"

Article Title: Heat shock protein 70 expression, keratin phosphorylation and Mallory body formation in hepatocytes from griseofulvin-intoxicated mice

Journal: Comparative Hepatology

doi: 10.1186/1476-5926-3-5

Distribution of keratin IFs and K18 pS33 in hepatocytes from control and GF-fed C3H mice. A, C, E, G keratin IFs; B, D, F, H K18 pS33; A, B) control; C, D) 2 week treatment; E, F) 6 week treatment; G, H) 5 month treatment. Asterisk in D shows an hepatocyte containing a high level of K18 pS33; arrow indicates a dilated bile canaliculi. Filled arrowheads in G and H indicate reactive MBs with Troma 1 and Ab8250 (anti-K18 pS33), respectively. Scale bar = 20 μm.
Figure Legend Snippet: Distribution of keratin IFs and K18 pS33 in hepatocytes from control and GF-fed C3H mice. A, C, E, G keratin IFs; B, D, F, H K18 pS33; A, B) control; C, D) 2 week treatment; E, F) 6 week treatment; G, H) 5 month treatment. Asterisk in D shows an hepatocyte containing a high level of K18 pS33; arrow indicates a dilated bile canaliculi. Filled arrowheads in G and H indicate reactive MBs with Troma 1 and Ab8250 (anti-K18 pS33), respectively. Scale bar = 20 μm.

Techniques Used: Mouse Assay

Biochemical analysis of livers from C3H and FVB/n mice. Western blots from C3H mouse livers; A, K8 and HSP70i; C, K8 pS79; E, K18 pS33; G, K8 pS436. Western blots from FVB/n mouse livers; B, K8; D, K8 pS79; F, K18 pS33.
Figure Legend Snippet: Biochemical analysis of livers from C3H and FVB/n mice. Western blots from C3H mouse livers; A, K8 and HSP70i; C, K8 pS79; E, K18 pS33; G, K8 pS436. Western blots from FVB/n mouse livers; B, K8; D, K8 pS79; F, K18 pS33.

Techniques Used: Mouse Assay, Western Blot

26) Product Images from "Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies"

Article Title: Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies

Journal: bioRxiv

doi: 10.1101/2020.06.02.131102

Proof-of-concept MInD-based SARS-CoV-2 specific antibody detection. IFCs were coated with commercial 2 μg·ml −1 SARS-CoV-2 spike protein peptide and blocked with BSA. A corresponding antibody was diluted either in PBS (black curves) or spiked in human serum (red curves) and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized magnetic particles. Assay time of this preliminary MInD setup was 42 minutes (without column preparation). Limit of detection (LOD) was determined by help of non-linear hill fit. Each data point represents mean ± SD (n = 2).
Figure Legend Snippet: Proof-of-concept MInD-based SARS-CoV-2 specific antibody detection. IFCs were coated with commercial 2 μg·ml −1 SARS-CoV-2 spike protein peptide and blocked with BSA. A corresponding antibody was diluted either in PBS (black curves) or spiked in human serum (red curves) and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized magnetic particles. Assay time of this preliminary MInD setup was 42 minutes (without column preparation). Limit of detection (LOD) was determined by help of non-linear hill fit. Each data point represents mean ± SD (n = 2).

Techniques Used:

Proof-of-concept MInD assay setup using IFC coated with SARS-CoV-2 antigen. Assay steps and assay time are indicated. IFCs were coated with commercial SARS-CoV-2 S-protein peptide and blocked with BSA. Corresponding antibody was diluted either in PBS or spiked in human serum and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized MNP. Finally, IFCs were inserted into the portable magnetic read-out device. Measuring signal can be correlated to the amount of antibody in the sample and antibody titer can be determined. Assay time of this preliminary MInD setup was 42 min which is approximately four times faster than ELISA (161 min).
Figure Legend Snippet: Proof-of-concept MInD assay setup using IFC coated with SARS-CoV-2 antigen. Assay steps and assay time are indicated. IFCs were coated with commercial SARS-CoV-2 S-protein peptide and blocked with BSA. Corresponding antibody was diluted either in PBS or spiked in human serum and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized MNP. Finally, IFCs were inserted into the portable magnetic read-out device. Measuring signal can be correlated to the amount of antibody in the sample and antibody titer can be determined. Assay time of this preliminary MInD setup was 42 min which is approximately four times faster than ELISA (161 min).

Techniques Used: Antigen Assay, Enzyme-linked Immunosorbent Assay

ELISA-based detection of SARS-CoV-2 spike protein specific antibody in PBS-buffer (black curve) or spiked in human serum (red curve). Antibody was diluted to concentrations ranging from 1.22 ng·ml −1 up to 5000 ng·ml −1 in each matrix and applied onto 2 ng·ml −1 SARS-CoV-2 spike protein peptide coated and BSA blocked microtiter plates. After addition of biotinylated secondary antibody, streptavidin-AP was applied. Limit of detection (LOD) was calculated using non-linear Hill fit (R2=0.997 for PBS-buffer and 0.996 in serum). Assay time of ELISA was 161 minutes. Each data point represents mean ± SD (n = 4 for PBS-buffer and n = 3 for serum).
Figure Legend Snippet: ELISA-based detection of SARS-CoV-2 spike protein specific antibody in PBS-buffer (black curve) or spiked in human serum (red curve). Antibody was diluted to concentrations ranging from 1.22 ng·ml −1 up to 5000 ng·ml −1 in each matrix and applied onto 2 ng·ml −1 SARS-CoV-2 spike protein peptide coated and BSA blocked microtiter plates. After addition of biotinylated secondary antibody, streptavidin-AP was applied. Limit of detection (LOD) was calculated using non-linear Hill fit (R2=0.997 for PBS-buffer and 0.996 in serum). Assay time of ELISA was 161 minutes. Each data point represents mean ± SD (n = 4 for PBS-buffer and n = 3 for serum).

Techniques Used: Enzyme-linked Immunosorbent Assay, Serum Assay

27) Product Images from "A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus"

Article Title: A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0136888

Both SP27 and MAP27 bind to anti-PGN mAb specifically. (A) The sequences of SP27 (Biotin- SA SPHHHSRLRSES GG ) and SP27’ (Biotin-SPHHHSRLRSES SAGG ). Underlined letters represent nonspecific flanking amino acids. (B) Both SP27 and SP27’ bind to anti-PGN mAb in a dose-dependent manner. (C) Both SP27 and SP27’ (100 μg/ml) specifically bind to anti-PGN mAb, but not to anti-LTA mAb. For ELISA assays in panel B and C, anti-PGN mAb or anti-LTA mAb was used to coat the wells at a concentration of 5 μg/ml, SP27, SP27’ or non-specific peptide L2 (in panel B, sequence: Biotin-HSGHWDFRQWWQPSGG) was then added at indicated concentrations and incubated at 37°C for 40 min, followed by detection with HRP-labeled streptavidin. (D) The structure diagram of MAP27. MAP27 was synthesized in a tetra-branched form that contains four copies of a sequence (SASPHHHSRLRSESGG) that mimics PGN epitope. (E) MAP27 binds to anti-PGN mAb specifically. (F) MAP27 binds to anti- S . aureus polyclonal antibodies specifically. For ELISA assays in panel E and F, MAP27 or MAPctrl was used to coat the wells of a microplate. Anti-PGN mAb or anti- S . aureus polyclonal antibodies was added, followed by detection with HRP-labeled antibodies. The absorbance was measured at OD 450nm . The results are shown as means ±SEM. * P
Figure Legend Snippet: Both SP27 and MAP27 bind to anti-PGN mAb specifically. (A) The sequences of SP27 (Biotin- SA SPHHHSRLRSES GG ) and SP27’ (Biotin-SPHHHSRLRSES SAGG ). Underlined letters represent nonspecific flanking amino acids. (B) Both SP27 and SP27’ bind to anti-PGN mAb in a dose-dependent manner. (C) Both SP27 and SP27’ (100 μg/ml) specifically bind to anti-PGN mAb, but not to anti-LTA mAb. For ELISA assays in panel B and C, anti-PGN mAb or anti-LTA mAb was used to coat the wells at a concentration of 5 μg/ml, SP27, SP27’ or non-specific peptide L2 (in panel B, sequence: Biotin-HSGHWDFRQWWQPSGG) was then added at indicated concentrations and incubated at 37°C for 40 min, followed by detection with HRP-labeled streptavidin. (D) The structure diagram of MAP27. MAP27 was synthesized in a tetra-branched form that contains four copies of a sequence (SASPHHHSRLRSESGG) that mimics PGN epitope. (E) MAP27 binds to anti-PGN mAb specifically. (F) MAP27 binds to anti- S . aureus polyclonal antibodies specifically. For ELISA assays in panel E and F, MAP27 or MAPctrl was used to coat the wells of a microplate. Anti-PGN mAb or anti- S . aureus polyclonal antibodies was added, followed by detection with HRP-labeled antibodies. The absorbance was measured at OD 450nm . The results are shown as means ±SEM. * P

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Sequencing, Incubation, Labeling, Synthesized

28) Product Images from "A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors"

Article Title: A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2018.07.001

Expression and Purification of Recombinant Target Proteins for Ribosome Display (A) Schematic drawing of GluA4-Fc and Fc, two recombinant proteins used as targets for ribosome display. The GluA4-Fc construct consists of the amino-terminal domain (ATD) of the glutamate receptor subunit 4 (GluA4) fused N-terminally to the Ig kappa chain signal peptide (SP) and C-terminally to the constant region of human IgG1 (huIgG1-Fc) for detection and purification and an Avi tag for biotinylation. As control in selections, only huIgG1-Fc with Avi tag were expressed (directly fused to the signal peptide). (B) Chromatograms of size exclusion chromatography (SEC) of proteins expressed in and purified from the cell culture supernatant of HEK293T cells via protein A. The calculated molecular weight of the corresponding peaks is indicated. (C) Reducing SDS-PAGE and western blot analysis of SEC-purified GluA4-Fc and Fc proteins produced in the absence (−) or presence of biotin (+) added to the culture. 2 μg and 20 ng of purified proteins were loaded onto 10% SDS gels, respectively. Purified proteins were visualized by PageBlue protein staining solution and detected by a huIgG1-Fc-specific antibody. Biotinylated proteins were detected using streptavidin-HRP.
Figure Legend Snippet: Expression and Purification of Recombinant Target Proteins for Ribosome Display (A) Schematic drawing of GluA4-Fc and Fc, two recombinant proteins used as targets for ribosome display. The GluA4-Fc construct consists of the amino-terminal domain (ATD) of the glutamate receptor subunit 4 (GluA4) fused N-terminally to the Ig kappa chain signal peptide (SP) and C-terminally to the constant region of human IgG1 (huIgG1-Fc) for detection and purification and an Avi tag for biotinylation. As control in selections, only huIgG1-Fc with Avi tag were expressed (directly fused to the signal peptide). (B) Chromatograms of size exclusion chromatography (SEC) of proteins expressed in and purified from the cell culture supernatant of HEK293T cells via protein A. The calculated molecular weight of the corresponding peaks is indicated. (C) Reducing SDS-PAGE and western blot analysis of SEC-purified GluA4-Fc and Fc proteins produced in the absence (−) or presence of biotin (+) added to the culture. 2 μg and 20 ng of purified proteins were loaded onto 10% SDS gels, respectively. Purified proteins were visualized by PageBlue protein staining solution and detected by a huIgG1-Fc-specific antibody. Biotinylated proteins were detected using streptavidin-HRP.

Techniques Used: Expressing, Purification, Recombinant, Construct, Size-exclusion Chromatography, Cell Culture, Molecular Weight, SDS Page, Western Blot, Produced, Staining

Workflow for the Selection of DARPins for Receptor-Targeted LVs and AAVs DARPin selection by ribosome display is shown in steps 1 and 2. All substeps of the ribosome display cycle are performed cell-free in vitro . Each cycle begins with the transcription and translation of a DARPin-encoding DNA library, flanked by a T7 promoter (T7), a ribosome binding site (RBS), and a spacer sequence. Ternary complexes of ribosome, the DARPin-encoding mRNA, and the translated polypeptide (shown in identical color) are formed and allowed to bind to the target protein during the selection process. The selection process encompasses three steps: pre-panning, counter-, and target selection. Pre-panning and counter-selection results in the elimination of the black and brown DARPins binding to the Fc domain or streptavidin used for immobilization of the target receptor. The green DARPin, in contrast, binds the target receptor and is carried forward to the next selection cycle. After the selection process, unbound complexes are washed away before elution, and reverse transcription of the mRNA is carried out. The cDNA fragments are PCR amplified and ligated to the upstream and downstream flanking sequences. The PCR-amplified ligation product is used as template library for the next ribosome display cycle or cloned into an expression vector for analysis of single clones on the protein level. After ribosome display, individual DARPin molecules are expressed as crude E. coli lysates (step 3), tested for their receptor binding ability (step 4), and subcloned into the corresponding viral vector plasmids (step 5) before small-scale generation of DARPin-displaying LV or AAV particles (step 6), which are finally analyzed for cell-type-specific gene transfer (step 7). Exemplarily an AAV vector is shown displaying five molecules of an individual DARPin clone on its surface, but the same procedure is applied for LV particles. Step 1 of the figure is adapted from Dreier and Plückthun. 53
Figure Legend Snippet: Workflow for the Selection of DARPins for Receptor-Targeted LVs and AAVs DARPin selection by ribosome display is shown in steps 1 and 2. All substeps of the ribosome display cycle are performed cell-free in vitro . Each cycle begins with the transcription and translation of a DARPin-encoding DNA library, flanked by a T7 promoter (T7), a ribosome binding site (RBS), and a spacer sequence. Ternary complexes of ribosome, the DARPin-encoding mRNA, and the translated polypeptide (shown in identical color) are formed and allowed to bind to the target protein during the selection process. The selection process encompasses three steps: pre-panning, counter-, and target selection. Pre-panning and counter-selection results in the elimination of the black and brown DARPins binding to the Fc domain or streptavidin used for immobilization of the target receptor. The green DARPin, in contrast, binds the target receptor and is carried forward to the next selection cycle. After the selection process, unbound complexes are washed away before elution, and reverse transcription of the mRNA is carried out. The cDNA fragments are PCR amplified and ligated to the upstream and downstream flanking sequences. The PCR-amplified ligation product is used as template library for the next ribosome display cycle or cloned into an expression vector for analysis of single clones on the protein level. After ribosome display, individual DARPin molecules are expressed as crude E. coli lysates (step 3), tested for their receptor binding ability (step 4), and subcloned into the corresponding viral vector plasmids (step 5) before small-scale generation of DARPin-displaying LV or AAV particles (step 6), which are finally analyzed for cell-type-specific gene transfer (step 7). Exemplarily an AAV vector is shown displaying five molecules of an individual DARPin clone on its surface, but the same procedure is applied for LV particles. Step 1 of the figure is adapted from Dreier and Plückthun. 53

Techniques Used: Selection, In Vitro, Binding Assay, Sequencing, Polymerase Chain Reaction, Amplification, Ligation, Clone Assay, Expressing, Plasmid Preparation

29) Product Images from "The Involvement of Proteoglycans in the Human Plasma Prekallikrein Interaction with the Cell Surface"

Article Title: The Involvement of Proteoglycans in the Human Plasma Prekallikrein Interaction with the Cell Surface

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091280

Endocytosis of biotin-prekallikrein by ECV304 cells. ECV304 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. They were treated with or without H-kininogen (100 nM, unlabelled) for 1 h and then treated with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and then treated with or without H-kininogen (100 nM, unlabelled) for 1 h and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by their colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by ECV304 cells at 37°C without H-kininogen (A–C): biotin-prekallikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekallikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by ECV304 at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).
Figure Legend Snippet: Endocytosis of biotin-prekallikrein by ECV304 cells. ECV304 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. They were treated with or without H-kininogen (100 nM, unlabelled) for 1 h and then treated with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and then treated with or without H-kininogen (100 nM, unlabelled) for 1 h and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by their colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by ECV304 cells at 37°C without H-kininogen (A–C): biotin-prekallikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekallikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by ECV304 at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy

Endocytosis of biotin-prekallikrein by CHO-745 cells. CHO-745 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. Then the cells were treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by CHO-745 cells at 37°C without H-kininogen (A–C): biotin-prekallikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekallikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by CHO-745 cells at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).
Figure Legend Snippet: Endocytosis of biotin-prekallikrein by CHO-745 cells. CHO-745 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. Then the cells were treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by CHO-745 cells at 37°C without H-kininogen (A–C): biotin-prekallikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekallikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by CHO-745 cells at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy

Endocytosis of biotin-prekallikrein by CHO-K1 cells. CHO-K1 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. They were then treated with or without H-kininogen (100 nM, unlabelled) for 30 min and then with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and then treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by their colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by CHO-K1 cells at 37°C without H-kininogen (A–C): biotin-prekalikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekalikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by CHO-K1 cells at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).
Figure Legend Snippet: Endocytosis of biotin-prekallikrein by CHO-K1 cells. CHO-K1 cells were grown on cover slips, and the lysosomes/endosomes were labeled with 0.5 μM LT Red DND-99 in incubation buffer for 20 min at 37°C. They were then treated with or without H-kininogen (100 nM, unlabelled) for 30 min and then with biotin-prekallikrein (100 nM) for 1 h at 37°C. The cells were incubated with FITC-conjugated streptavidin (green). Alternatively, after labeling with 0.5 μM LT Red DND-99 at 37°C, the cells were maintained at 4°C and then treated with or without H-kininogen (100 nM, unlabelled) for 30 min and with biotin-prekallikrein (100 nM) for 1 h at 4°C. Biotin-prekallikrein (green) endocytosis and intracellular localization are indicated by their colocalization with acidic vesicles previously labeled with LT Red DND-99 (red) and were analyzed by confocal fluorescence microscopy. Normal endocytosis by CHO-K1 cells at 37°C without H-kininogen (A–C): biotin-prekalikrein (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); with H-kininogen (D–F): biotin-prekalikrein (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). Normal endocytosis by CHO-K1 cells at 4°C without H-kininogen (G–I): biotin-prekallikrein (G), lysosomes/endosomes labeled with LT Red DND-99 (H), merged images and diphasic contrast (I); with H-kininogen (J–L): biotin-prekallikrein (J), lysosomes/endosomes labeled with LT Red DND-99 (K), merged images and diphasic contrast (L).

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy

30) Product Images from "Autotaxin, a lysophosphatidic acid-producing ectoenzyme, promotes lymphocyte entry into secondary lymphoid organs"

Article Title: Autotaxin, a lysophosphatidic acid-producing ectoenzyme, promotes lymphocyte entry into secondary lymphoid organs

Journal: Nature immunology

doi: 10.1038/ni1573

Secretion of ATX and HECs and transfected MDCK cells. ( a ) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit IgG as a control. The results shown
Figure Legend Snippet: Secretion of ATX and HECs and transfected MDCK cells. ( a ) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit IgG as a control. The results shown

Techniques Used: Transfection, Isolation, SDS Page

31) Product Images from "The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis"

Article Title: The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky001

Purified Esco1 acetylates α-tubulin peptide in vitro . ( A ) Purification of Flag-tagged Esco1 proteins. Esco1 and enzymatically mutant Esco1-G768D were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco1-Flag and Esco1-G768D-Flag were identified by coomassie staining and western blotting using anti-Esco1 antibody. ( B ) The synthesized peptide of α-tubulin was identified by western blotting using Streptavidin-HRP. ( C ) In vitro acetylation assay with purified Esco1 proteins and synthesized peptide. Synthesized α-tubulin peptide was incubated with or without purified Esco1-Flag, Esco1-G768D-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-acetyl-α-tubulin (Lys 40) antibody for acetylation levels of α-tubulin and Streptavidin-HRP as a loading control.
Figure Legend Snippet: Purified Esco1 acetylates α-tubulin peptide in vitro . ( A ) Purification of Flag-tagged Esco1 proteins. Esco1 and enzymatically mutant Esco1-G768D were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco1-Flag and Esco1-G768D-Flag were identified by coomassie staining and western blotting using anti-Esco1 antibody. ( B ) The synthesized peptide of α-tubulin was identified by western blotting using Streptavidin-HRP. ( C ) In vitro acetylation assay with purified Esco1 proteins and synthesized peptide. Synthesized α-tubulin peptide was incubated with or without purified Esco1-Flag, Esco1-G768D-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-acetyl-α-tubulin (Lys 40) antibody for acetylation levels of α-tubulin and Streptavidin-HRP as a loading control.

Techniques Used: Purification, In Vitro, Mutagenesis, Staining, Western Blot, Synthesized, Acetylation Assay, Incubation

32) Product Images from "Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin"

Article Title: Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.0209053

Intracellular CRAMP expression in blood and exudate PMN. (A) Western blot analysis of bPMN lysates using affinity-purified rabbit anti-CRAMP antibody. (B) Intracellular staining of pPMN with rabbit anti-CRAMP antibody followed by Cy3-conjugated donkey anti-rabbit antibody (i) and identical staining performed with antibody preincubated with excess of synthetic CRAMP peptide (ii). (C, Upper) PMN purified from blood (i), peritoneal exudate (ii), and TCF (iii) from C57BL/6 mice were stained intracellularly with biotinylated rabbit anti-CRAMP (black lines) or isotype control (gray lines) antibody, followed by RPE-conjugated Streptavidin and analyzed by flow cytometry. (iv) Purified TCF-PMN stained with the neutrophil marker NIMP-R14 (black line) or isotype control (gray line) antibody followed by FITC-conjugated goat anti-rat antibody. Graphs are representative of two to five independent experiments. (C, Lower) bPMN (i), pPMN (ii), and TCF-PMN (iii) were immunolabeled with rabbit anti-CRAMP or isotype control (not shown) antibody, followed by Cy3-conjugated donkey anti-rabbit antibody and examined by confocal microscopy. Isotype controls showed no detectable staining. Fluorescence micrographs (original magnification, ×100) are representatives of three independent experiments. FL 2, Fluorescence 2. (D) Total intracellular CRAMP in lysates of bPMN and pPMN of at least three C57BL/6 mice was determined by ELISA.
Figure Legend Snippet: Intracellular CRAMP expression in blood and exudate PMN. (A) Western blot analysis of bPMN lysates using affinity-purified rabbit anti-CRAMP antibody. (B) Intracellular staining of pPMN with rabbit anti-CRAMP antibody followed by Cy3-conjugated donkey anti-rabbit antibody (i) and identical staining performed with antibody preincubated with excess of synthetic CRAMP peptide (ii). (C, Upper) PMN purified from blood (i), peritoneal exudate (ii), and TCF (iii) from C57BL/6 mice were stained intracellularly with biotinylated rabbit anti-CRAMP (black lines) or isotype control (gray lines) antibody, followed by RPE-conjugated Streptavidin and analyzed by flow cytometry. (iv) Purified TCF-PMN stained with the neutrophil marker NIMP-R14 (black line) or isotype control (gray line) antibody followed by FITC-conjugated goat anti-rat antibody. Graphs are representative of two to five independent experiments. (C, Lower) bPMN (i), pPMN (ii), and TCF-PMN (iii) were immunolabeled with rabbit anti-CRAMP or isotype control (not shown) antibody, followed by Cy3-conjugated donkey anti-rabbit antibody and examined by confocal microscopy. Isotype controls showed no detectable staining. Fluorescence micrographs (original magnification, ×100) are representatives of three independent experiments. FL 2, Fluorescence 2. (D) Total intracellular CRAMP in lysates of bPMN and pPMN of at least three C57BL/6 mice was determined by ELISA.

Techniques Used: Expressing, Western Blot, Affinity Purification, Staining, Purification, Mouse Assay, Flow Cytometry, Cytometry, Marker, Immunolabeling, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay

33) Product Images from "Characterization of Rabbit CD5 Isoforms"

Article Title: Characterization of Rabbit CD5 Isoforms

Journal: Molecular immunology

doi: 10.1016/j.molimm.2009.05.026

Flow cytometric analyses. A. Thymocytes and appendix cells from 2-week-old rabbits stained with KEN-5 mAb and polyclonal goat anti-rabbit IgM. Percentages of KEN-5 + , IgM + or double positive cells are indicated. B. Inhibition of KEN-5 staining by polyclonal goat anti-CD5 (5-week-old rabbit). Left panels, appendix (APP). In the presence of control goat IgG (g-IgG), KEN-5 stains CD4 + cells (4%) identified with mAb KEN-4 (top) and goat anti-CD5 blocks this staining (bottom). Right panels, spleen (SPL). KEN-5 stains IgM - spleen cells (32%) (top). Goat anti-rabbit CD5 antibody blocks the staining without substantially interfering with IgM staining (bottom). Percentages of KEN-5 + , CD4 + or double positive cells in appendix and of KEN-5 + , IgM + or double positive cells in spleen are indicated.
Figure Legend Snippet: Flow cytometric analyses. A. Thymocytes and appendix cells from 2-week-old rabbits stained with KEN-5 mAb and polyclonal goat anti-rabbit IgM. Percentages of KEN-5 + , IgM + or double positive cells are indicated. B. Inhibition of KEN-5 staining by polyclonal goat anti-CD5 (5-week-old rabbit). Left panels, appendix (APP). In the presence of control goat IgG (g-IgG), KEN-5 stains CD4 + cells (4%) identified with mAb KEN-4 (top) and goat anti-CD5 blocks this staining (bottom). Right panels, spleen (SPL). KEN-5 stains IgM - spleen cells (32%) (top). Goat anti-rabbit CD5 antibody blocks the staining without substantially interfering with IgM staining (bottom). Percentages of KEN-5 + , CD4 + or double positive cells in appendix and of KEN-5 + , IgM + or double positive cells in spleen are indicated.

Techniques Used: Flow Cytometry, Staining, Inhibition

Immunofluorescence and confocal imaging. A. Staining of appendix sections with polyclonal biotin-conjugated goat anti-IgM followed by streptavidin Alexa Fluor 647 conjugate (white pseudocolor) and IgG1 KEN-5-FITC followed by goat anti-mouse IgG1-FITC (green) and finally by DAPI to identify nuclei (blue). Scale bar 100 μm. B. Staining of a rabbit spleen section with polyclonal biotin-conjugated goat anti-IgM followed by streptavidin Alexa Fluor 647 conjugate (white pseudocolor), IgG1 KEN-5-FITC followed by goat anti-mouse IgG1-FITC (green) and mouse anti-CD5 mAb (anti-D2 peptide 5A7) followed by Alexa Fluor 568 goat anti-mouse IgG2a (red), and finally by DAPI to identify nuclei (blue). Scale bar 20 μm (left). Scale bar in magnified image of area enclosed in white box (right)10 μm.
Figure Legend Snippet: Immunofluorescence and confocal imaging. A. Staining of appendix sections with polyclonal biotin-conjugated goat anti-IgM followed by streptavidin Alexa Fluor 647 conjugate (white pseudocolor) and IgG1 KEN-5-FITC followed by goat anti-mouse IgG1-FITC (green) and finally by DAPI to identify nuclei (blue). Scale bar 100 μm. B. Staining of a rabbit spleen section with polyclonal biotin-conjugated goat anti-IgM followed by streptavidin Alexa Fluor 647 conjugate (white pseudocolor), IgG1 KEN-5-FITC followed by goat anti-mouse IgG1-FITC (green) and mouse anti-CD5 mAb (anti-D2 peptide 5A7) followed by Alexa Fluor 568 goat anti-mouse IgG2a (red), and finally by DAPI to identify nuclei (blue). Scale bar 20 μm (left). Scale bar in magnified image of area enclosed in white box (right)10 μm.

Techniques Used: Immunofluorescence, Imaging, Staining

34) Product Images from "Activated protein C inhibits LPS-mediated acetylation and secretion of HMGB1 in endothelial cells"

Article Title: Activated protein C inhibits LPS-mediated acetylation and secretion of HMGB1 in endothelial cells

Journal: Journal of thrombosis and haemostasis : JTH

doi: 10.1111/jth.14425

APC inhibits LPS-mediated HMGB1 lysosomal localization in endothelial cells. (A) EA.hy926 ells were pretreated with APC (20 nM for 3h) followed by stimulation with LPS (1 µg/mL for 1h). Cells were then fixed and permeabilized and HMGB1 was stained with rabbit anti-HMGB1 and Cy3-conjugated goat anti-rabbit IgG. LAMP1, a lysosome marker, was stained with mouse anti-LAMP1 antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. (B) The same as panel A except that HMGB1 was stained employing rabbit anti-acetylated HMGB1 (Ace-HMGB1) and Cy3-conjugated goat anti-rabbit IgG. Arrows indicate colocalization of HMGB1 or acetylated-HMGB1 with LAMP1. The magnified insets correspond to the cells from LPS and APC+LPS groups. Scale bar: 10 μm.
Figure Legend Snippet: APC inhibits LPS-mediated HMGB1 lysosomal localization in endothelial cells. (A) EA.hy926 ells were pretreated with APC (20 nM for 3h) followed by stimulation with LPS (1 µg/mL for 1h). Cells were then fixed and permeabilized and HMGB1 was stained with rabbit anti-HMGB1 and Cy3-conjugated goat anti-rabbit IgG. LAMP1, a lysosome marker, was stained with mouse anti-LAMP1 antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. (B) The same as panel A except that HMGB1 was stained employing rabbit anti-acetylated HMGB1 (Ace-HMGB1) and Cy3-conjugated goat anti-rabbit IgG. Arrows indicate colocalization of HMGB1 or acetylated-HMGB1 with LAMP1. The magnified insets correspond to the cells from LPS and APC+LPS groups. Scale bar: 10 μm.

Techniques Used: Staining, Marker, Immunofluorescence, Confocal Microscopy

APC inhibits LPS-mediated HMGB1 translocation and lysosomal localization in macrophages. J774A.1 macrophages were pretreated with APC (20 nM for 3h) followed by stimulation with LPS (1 µg/mL for 1h). Cells were then fixed, permeabilized and HMGB1 was stained with rabbit anti-HMGB1 antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Quantification of translocated cells is shown on the right. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. (B) The same as panel A except that HMGB1 was stained with rabbit anti-HMGB1 followed by Cy3-conjugated goat anti-rabbit IgG. LAMP1 was stained with mouse anti-LAMP1 antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate colocalization of HMGB1 with LAMP1. The magnified insets correspond to the cells from LPS and APC+LPS groups. Scale bar: 10 μm. Results are shown as means ± SE. *p
Figure Legend Snippet: APC inhibits LPS-mediated HMGB1 translocation and lysosomal localization in macrophages. J774A.1 macrophages were pretreated with APC (20 nM for 3h) followed by stimulation with LPS (1 µg/mL for 1h). Cells were then fixed, permeabilized and HMGB1 was stained with rabbit anti-HMGB1 antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Quantification of translocated cells is shown on the right. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. (B) The same as panel A except that HMGB1 was stained with rabbit anti-HMGB1 followed by Cy3-conjugated goat anti-rabbit IgG. LAMP1 was stained with mouse anti-LAMP1 antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate colocalization of HMGB1 with LAMP1. The magnified insets correspond to the cells from LPS and APC+LPS groups. Scale bar: 10 μm. Results are shown as means ± SE. *p

Techniques Used: Translocation Assay, Staining, Immunofluorescence, Confocal Microscopy

APC inhibits LPS-mediated HMGB1 expression and cytoplasmic translocation in mouse cremaster muscle. (A) Mice were intrascrotally injected with APC or APC-2Cys (0.2 mg/kg) followed by LPS injection (0.5 mg/kg). After 1 hour, tissue was harvested for lysis or histological process. Tissue lysates were immunoblotted for HMGB1 and β-actin. (B) Cryosection of cremaster muscle tissue were fixed and permeabilized. HMGB1 was stained with biotin-conjugated rabbit anti-HMGB1 antibody and Cy3-conjugated streptavidin; CD31 was stained with rat anti-CD31 antibody and Alexa Fluor 488-conjugated donkey anti-rat IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. Scale bar: 10 μm. Results are shown as means ± SE. *p
Figure Legend Snippet: APC inhibits LPS-mediated HMGB1 expression and cytoplasmic translocation in mouse cremaster muscle. (A) Mice were intrascrotally injected with APC or APC-2Cys (0.2 mg/kg) followed by LPS injection (0.5 mg/kg). After 1 hour, tissue was harvested for lysis or histological process. Tissue lysates were immunoblotted for HMGB1 and β-actin. (B) Cryosection of cremaster muscle tissue were fixed and permeabilized. HMGB1 was stained with biotin-conjugated rabbit anti-HMGB1 antibody and Cy3-conjugated streptavidin; CD31 was stained with rat anti-CD31 antibody and Alexa Fluor 488-conjugated donkey anti-rat IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. Scale bar: 10 μm. Results are shown as means ± SE. *p

Techniques Used: Expressing, Translocation Assay, Mouse Assay, Injection, Lysis, Staining, Immunofluorescence, Confocal Microscopy

Mac-1 and PAR1 but not EPCR is required for APC inhibition of HMGB1 translocation and lysosomal localization in macrophages. (A) J774A.1 macrophages were pretreated with Mac-1, EPCR or PAR1 function-blocking antibody (15–20 µg/mL for 1h) followed by treatment with APC (20 nM for 3h) before stimulation with LPS (1 µg/mL for 1h). Cells were then fixed, permeabilized and HMGB1 was stained with rabbit anti-HMGB1 antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. (B) The same as panel A except that HMGB1 was stained with rabbit anti-HMGB1 followed by Cy3-conjugated goat anti-rabbit IgG. LAMP1 was stained with mouse anti-LAMP1 antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate colocalization of HMGB1 with LAMP1. Scale bar: 10 μm (A and B).
Figure Legend Snippet: Mac-1 and PAR1 but not EPCR is required for APC inhibition of HMGB1 translocation and lysosomal localization in macrophages. (A) J774A.1 macrophages were pretreated with Mac-1, EPCR or PAR1 function-blocking antibody (15–20 µg/mL for 1h) followed by treatment with APC (20 nM for 3h) before stimulation with LPS (1 µg/mL for 1h). Cells were then fixed, permeabilized and HMGB1 was stained with rabbit anti-HMGB1 antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. (B) The same as panel A except that HMGB1 was stained with rabbit anti-HMGB1 followed by Cy3-conjugated goat anti-rabbit IgG. LAMP1 was stained with mouse anti-LAMP1 antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate colocalization of HMGB1 with LAMP1. Scale bar: 10 μm (A and B).

Techniques Used: Inhibition, Translocation Assay, Blocking Assay, Staining, Immunofluorescence, Confocal Microscopy

35) Product Images from "Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands"

Article Title: Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI89535

Ccl19-Cre –mediated Dll1 and Dll4 inactivation does not impair T cell recruitment or proliferation in SLOs after irradiation, but selectively affects Notch target gene transcripts. ( A – E ) Absolute numbers ( A ), proliferation (CFSE dilution) on day 2.5 ( B ) versus day 6 after transplantation ( C ), and expression of activation markers. Bars in histograms define gating for proliferated CFSE low and unproliferated CFSE hi cells, and numbers indicate the percentage of gated cells among parental cell populations, as identified by flow cytometric analysis. Bar graphs represent the mean percentage of proliferated (CFSE low ) cells in each population ( n =5 mice/group); error bars indicate SD. ( D and E ) by donor-derived CD4 + and CD8 + T cells after transplantation into lethally irradiated (12 Gy) littermate control Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ B6 recipient mice. Donor cells were isolated on day 2.5 or day 6 after transplantation ( n = 5 mice/group). ( F ) Abundance of the indicated transcripts (qRT-PCR) in sort-purified donor-derived CFSE diluted BALB/c CD4 + T cells on day 2 after transplantation into irradiated B6 hosts treated with or without anti-DLL1/4 antibodies. * P
Figure Legend Snippet: Ccl19-Cre –mediated Dll1 and Dll4 inactivation does not impair T cell recruitment or proliferation in SLOs after irradiation, but selectively affects Notch target gene transcripts. ( A – E ) Absolute numbers ( A ), proliferation (CFSE dilution) on day 2.5 ( B ) versus day 6 after transplantation ( C ), and expression of activation markers. Bars in histograms define gating for proliferated CFSE low and unproliferated CFSE hi cells, and numbers indicate the percentage of gated cells among parental cell populations, as identified by flow cytometric analysis. Bar graphs represent the mean percentage of proliferated (CFSE low ) cells in each population ( n =5 mice/group); error bars indicate SD. ( D and E ) by donor-derived CD4 + and CD8 + T cells after transplantation into lethally irradiated (12 Gy) littermate control Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ B6 recipient mice. Donor cells were isolated on day 2.5 or day 6 after transplantation ( n = 5 mice/group). ( F ) Abundance of the indicated transcripts (qRT-PCR) in sort-purified donor-derived CFSE diluted BALB/c CD4 + T cells on day 2 after transplantation into irradiated B6 hosts treated with or without anti-DLL1/4 antibodies. * P

Techniques Used: Irradiation, Transplantation Assay, Expressing, Activation Assay, Flow Cytometry, Mouse Assay, Derivative Assay, Isolation, Quantitative RT-PCR, Purification

Fibroblastic niches in spleen express DLL1/4 Notch ligands and localize next to alloreactive T cells. ( A and B ) Immunofluorescence microscopy of splenic cryosections from Tg Ccl19-Cre+ Dll1 +/+ Dll4 +/+ ROSA26 eYFP and Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ ROSA26 eYFP mice stained for GFP, CD35, and DLL4 ( A ) or GFP, CD157, and DLL4 ( B ). ( C – E ) Immunofluorescence microscopy of splenic cryosections from lethally irradiated (8.5 Gy) BALB/c mice transplanted with CMTMR-labeled alloantigen-specific CD4 + 4C TCR–transgenic cells and pulsed with EdU 12 hours prior to organ collection to label proliferating cells. Cryosections were incubated with Alexa Fluor 488 picolyl azide to reveal EdU, along with anti-DLL4 ( C ), anti-CD35 ( D ), or anti-CD157 ( E ). Organs were collected on day 1.5 after transplantation. Data are representative of at least 2 experiments.
Figure Legend Snippet: Fibroblastic niches in spleen express DLL1/4 Notch ligands and localize next to alloreactive T cells. ( A and B ) Immunofluorescence microscopy of splenic cryosections from Tg Ccl19-Cre+ Dll1 +/+ Dll4 +/+ ROSA26 eYFP and Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ ROSA26 eYFP mice stained for GFP, CD35, and DLL4 ( A ) or GFP, CD157, and DLL4 ( B ). ( C – E ) Immunofluorescence microscopy of splenic cryosections from lethally irradiated (8.5 Gy) BALB/c mice transplanted with CMTMR-labeled alloantigen-specific CD4 + 4C TCR–transgenic cells and pulsed with EdU 12 hours prior to organ collection to label proliferating cells. Cryosections were incubated with Alexa Fluor 488 picolyl azide to reveal EdU, along with anti-DLL4 ( C ), anti-CD35 ( D ), or anti-CD157 ( E ). Organs were collected on day 1.5 after transplantation. Data are representative of at least 2 experiments.

Techniques Used: Immunofluorescence, Microscopy, Mouse Assay, Staining, Irradiation, Labeling, Transgenic Assay, Incubation, Transplantation Assay

DLL4 expression within different LN nonhematopoietic cell populations after allogeneic BMT. ( A ) LN stromal cell subsets. Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ and Tg Ccl19-Cre– littermate control mice were lethally irradiated before infusion of allogeneic BALB/c splenocytes. LNs were collected on day 1.5 after transplantation and enzymatically digested. Dot plots show representative flow cytometric analysis of CD45 – Ter119 – nonhematopoietic stromal cells. ( B ) DLL4 expression in LN-resident nonhematopoietic cell subsets. Roman numerals refer to the cell populations shown in A . ( C ) MFI comparing DLL4 expression in stromal cell subsets from Tg Ccl19-Cre– versus Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice as well as isotype staining in controls. ** P
Figure Legend Snippet: DLL4 expression within different LN nonhematopoietic cell populations after allogeneic BMT. ( A ) LN stromal cell subsets. Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ and Tg Ccl19-Cre– littermate control mice were lethally irradiated before infusion of allogeneic BALB/c splenocytes. LNs were collected on day 1.5 after transplantation and enzymatically digested. Dot plots show representative flow cytometric analysis of CD45 – Ter119 – nonhematopoietic stromal cells. ( B ) DLL4 expression in LN-resident nonhematopoietic cell subsets. Roman numerals refer to the cell populations shown in A . ( C ) MFI comparing DLL4 expression in stromal cell subsets from Tg Ccl19-Cre– versus Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice as well as isotype staining in controls. ** P

Techniques Used: Expressing, Mouse Assay, Irradiation, Transplantation Assay, Flow Cytometry, Staining

Host hematopoietic cells are dispensable as cellular sources of DLL1/4 Notch ligands in acute GVHD. ( A ) Experimental strategy. BM chimeras were generated via transplantation of syngeneic B6-CD45.2 + poly(I:C)-induced Tg Mx1-Cre– littermate control or Tg Mx1-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ BM into irradiated B6-CD45.1 recipients. After reestablishment of steady-state hematopoiesis 12 weeks later, BM chimeras were subjected to a second syngeneic or allogeneic transplant, with or without systemic anti-DLL1/4 blockade. ( B ) Quantification of Dll1 and Dll4 inactivation in sort-purified Gr1 + CD11b + blood myeloid cells from BM chimeras 12 weeks after transplantation (PCR). In this particular experiment, control BM chimeras were generated from poly(I:C)-induced Tg Mx1-Cre– Dll1 fl/+ Dll4 fl/+ donor mice. Each lane represents an individual mouse. ( C ) Donor chimerism (frequency of CD45.2 + donor cells) in the indicated splenic cell populations 12 weeks after transplantation. MΦ, macrophages; pDCs, plasmacytoid DCs. ( D ) Survival and weight loss of lethally irradiated (11 Gy) BM chimeras transplanted with 8 × 10 6 TCD BM plus 30 × 10 6 B6 splenocytes (syngeneic control) or 30 × 10 6 allogeneic BALB/c splenocytes (allo-BMT). Isotype control or anti-DLL1/4 antibodies were injected i.p. on days 0, 3, 7, and 10 ( n = 10 mice/group). ( E ) Abundance of Dtx1 Notch target gene transcripts (qRT-PCR) in sort-purified donor CD4 + T cells and CD8 + cells on day 6 ( n = 5 mice/group). * P
Figure Legend Snippet: Host hematopoietic cells are dispensable as cellular sources of DLL1/4 Notch ligands in acute GVHD. ( A ) Experimental strategy. BM chimeras were generated via transplantation of syngeneic B6-CD45.2 + poly(I:C)-induced Tg Mx1-Cre– littermate control or Tg Mx1-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ BM into irradiated B6-CD45.1 recipients. After reestablishment of steady-state hematopoiesis 12 weeks later, BM chimeras were subjected to a second syngeneic or allogeneic transplant, with or without systemic anti-DLL1/4 blockade. ( B ) Quantification of Dll1 and Dll4 inactivation in sort-purified Gr1 + CD11b + blood myeloid cells from BM chimeras 12 weeks after transplantation (PCR). In this particular experiment, control BM chimeras were generated from poly(I:C)-induced Tg Mx1-Cre– Dll1 fl/+ Dll4 fl/+ donor mice. Each lane represents an individual mouse. ( C ) Donor chimerism (frequency of CD45.2 + donor cells) in the indicated splenic cell populations 12 weeks after transplantation. MΦ, macrophages; pDCs, plasmacytoid DCs. ( D ) Survival and weight loss of lethally irradiated (11 Gy) BM chimeras transplanted with 8 × 10 6 TCD BM plus 30 × 10 6 B6 splenocytes (syngeneic control) or 30 × 10 6 allogeneic BALB/c splenocytes (allo-BMT). Isotype control or anti-DLL1/4 antibodies were injected i.p. on days 0, 3, 7, and 10 ( n = 10 mice/group). ( E ) Abundance of Dtx1 Notch target gene transcripts (qRT-PCR) in sort-purified donor CD4 + T cells and CD8 + cells on day 6 ( n = 5 mice/group). * P

Techniques Used: Generated, Transplantation Assay, Irradiation, Purification, Polymerase Chain Reaction, Mouse Assay, Injection, Quantitative RT-PCR

Ccl19-Cre + lineage–traced stromal cells are the critical cellular source of DLL1/4 Notch ligands during acute GVHD. ( A ) LNs were collected on day 1.5 after transplantation from lethally irradiated Tg Ccl19-Cre+ ROSA26 eYFP mice receiving allogeneic BALB/c splenocytes and enzymatically digested. ( A , top) eYFP expression in LN-resident bulk fibroblastic stromal cells (PDPN + CD31 – ) as well as subfractionated CD157 + FRCs, CD157 – FRCs, and CD21 + FDCs. ( A , middle) eYFP in LECs, BECs, PDPN – CD31 – stromal cells (DNs). ( A , bottom) eYFP in macrophages, conventional DCs (cDCs), pDCs, and skin-derived Langerhans cells. Bars in histograms define gating for eYFP + cells, and numbers indicate the percentage of gated eYFP + cells within parental cell populations, as identified by flow cytometric analysis. Bar graph in A shows the mean percentage of eYFP expression in each indicated nonhematopoietic subset ( n = 4 mice/group; error bars indicate SD). ( B ) Survival, GVHD score, and weight of lethally irradiated (12 Gy) littermate control Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice that were transplanted with 10 × 10 6 TCD BM only or 10 × 10 6 TCD BM plus 20 × 10 6 allogeneic BALB/c splenocytes. Isotype control or anti-DLL1/4–neutralizing antibodies were injected i.p. on days 0, 3, 7, and 10 ( n = 10 mice/group). ( C ) Intracellular cytokines in donor CD4 + cells after anti-CD3/CD28 restimulation on day 6 ( n = 5 mice/group). ( D ) Relative abundance of Dtx1 and Hes1 Notch target gene transcripts in donor CD4 + T cells sort purified from Tg Ccl19-Cre– plus isotype control, Tg Ccl19-Cre– plus anti-DLL1/4, or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ recipient mice on day 2 after transplantation ( n = 5 mice/group). * P
Figure Legend Snippet: Ccl19-Cre + lineage–traced stromal cells are the critical cellular source of DLL1/4 Notch ligands during acute GVHD. ( A ) LNs were collected on day 1.5 after transplantation from lethally irradiated Tg Ccl19-Cre+ ROSA26 eYFP mice receiving allogeneic BALB/c splenocytes and enzymatically digested. ( A , top) eYFP expression in LN-resident bulk fibroblastic stromal cells (PDPN + CD31 – ) as well as subfractionated CD157 + FRCs, CD157 – FRCs, and CD21 + FDCs. ( A , middle) eYFP in LECs, BECs, PDPN – CD31 – stromal cells (DNs). ( A , bottom) eYFP in macrophages, conventional DCs (cDCs), pDCs, and skin-derived Langerhans cells. Bars in histograms define gating for eYFP + cells, and numbers indicate the percentage of gated eYFP + cells within parental cell populations, as identified by flow cytometric analysis. Bar graph in A shows the mean percentage of eYFP expression in each indicated nonhematopoietic subset ( n = 4 mice/group; error bars indicate SD). ( B ) Survival, GVHD score, and weight of lethally irradiated (12 Gy) littermate control Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice that were transplanted with 10 × 10 6 TCD BM only or 10 × 10 6 TCD BM plus 20 × 10 6 allogeneic BALB/c splenocytes. Isotype control or anti-DLL1/4–neutralizing antibodies were injected i.p. on days 0, 3, 7, and 10 ( n = 10 mice/group). ( C ) Intracellular cytokines in donor CD4 + cells after anti-CD3/CD28 restimulation on day 6 ( n = 5 mice/group). ( D ) Relative abundance of Dtx1 and Hes1 Notch target gene transcripts in donor CD4 + T cells sort purified from Tg Ccl19-Cre– plus isotype control, Tg Ccl19-Cre– plus anti-DLL1/4, or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ recipient mice on day 2 after transplantation ( n = 5 mice/group). * P

Techniques Used: Transplantation Assay, Irradiation, Mouse Assay, Expressing, Derivative Assay, Flow Cytometry, Injection, Purification

Ccl19-Cre –mediated Dll1 and Dll4 inactivation preserves lymphocyte numbers and distribution in SLOs at steady state. ( A ) Absolute numbers of CD4 + T cells, CD8 + T cells, and B cells in spleens and LNs of B6 littermate control Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice at steady state ( n = 5 mice/group). ( B and C ) CD62L and CD44 expression in CD4 + ( B ) and CD8 + ( C ) T cells from spleens and LNs of Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice at steady state. ( D ) Abundance of Il7 transcripts (qRT-PCR) in sort-purified PDPN + CD31 – fibroblastic stromal cells from Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice ( n = 5 mice/group). NS = P > 0.05, by unpaired, 2-tailed Student’s t test. Data are representative of at least 3 experiments; error bars indicate SD.
Figure Legend Snippet: Ccl19-Cre –mediated Dll1 and Dll4 inactivation preserves lymphocyte numbers and distribution in SLOs at steady state. ( A ) Absolute numbers of CD4 + T cells, CD8 + T cells, and B cells in spleens and LNs of B6 littermate control Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice at steady state ( n = 5 mice/group). ( B and C ) CD62L and CD44 expression in CD4 + ( B ) and CD8 + ( C ) T cells from spleens and LNs of Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice at steady state. ( D ) Abundance of Il7 transcripts (qRT-PCR) in sort-purified PDPN + CD31 – fibroblastic stromal cells from Tg Ccl19-Cre– or Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ mice ( n = 5 mice/group). NS = P > 0.05, by unpaired, 2-tailed Student’s t test. Data are representative of at least 3 experiments; error bars indicate SD.

Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Purification

An early pulse of Notch signaling is critical to drive pathogenic T cell alloreactivity after BMT. ( A ) Dosing schedule of systemic neutralizing antibodies against DLL1 and DLL4 Notch ligands. ( B ) Survival, GVHD score, and weight of lethally irradiated (8.5 Gy) BALB/c mice transplanted with 5 × 10 6 T cell–depleted (TCD) B6 BM or 5 × 10 6 TCD B6 BM plus 5 × 10 6 allogeneic B6 splenocytes. Isotype control versus anti-DLL1/4 antibodies were injected i.p., as shown in A ( n = 10 mice/group). ( C ) Intracellular cytokine production by donor CD4 + T cells after anti-CD3/CD28 restimulation on day 6 after transplantation ( n = 5 mice/group). ( D ) Intracellular FoxP3 in donor CD4 + T cells on day 6 ( n = 5 mice/group). * P
Figure Legend Snippet: An early pulse of Notch signaling is critical to drive pathogenic T cell alloreactivity after BMT. ( A ) Dosing schedule of systemic neutralizing antibodies against DLL1 and DLL4 Notch ligands. ( B ) Survival, GVHD score, and weight of lethally irradiated (8.5 Gy) BALB/c mice transplanted with 5 × 10 6 T cell–depleted (TCD) B6 BM or 5 × 10 6 TCD B6 BM plus 5 × 10 6 allogeneic B6 splenocytes. Isotype control versus anti-DLL1/4 antibodies were injected i.p., as shown in A ( n = 10 mice/group). ( C ) Intracellular cytokine production by donor CD4 + T cells after anti-CD3/CD28 restimulation on day 6 after transplantation ( n = 5 mice/group). ( D ) Intracellular FoxP3 in donor CD4 + T cells on day 6 ( n = 5 mice/group). * P

Techniques Used: Irradiation, Mouse Assay, Injection, Transplantation Assay

36) Product Images from "Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid"

Article Title: Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0577-8

Axonal damage and loss in the spinal cord and optic nerve of the NMO-rat. a Axon injury detected in the NMO-rat compared to the Control-rat (rats infused with IgG AQP4+ 2 and IgG Control 2, D7) using neurofilament immunostaining: reduced number of axons detected as NF-M-positive spots in the white matter (WM); fragmentation and reduced axon thickness in the gray matter (GM). b Classification (10–20 to 100–140 μm 2 , ImageJ) and quantification (mean by field) of NF-M-positive spots in the spinal cord of the NMO-rats ( n = 6) compared to the Control-rats ( n = 6): loss of axons with 60–140 μm 2 sections in the NMO-rats (in cart: evaluation of the total axon number, p = 0.03). c Co-detection of myelin alteration (MBP in green ) and axonal loss (neurofilament NF-M subtype in red ) in the spinal cord of the NMO-rat compared to the Control-rat. d Increased expression of the NF-H phosphorylated form, a marker of axon injury, detected by Western blot (pNF-H/NF-H ratio; p = 0.04). e Axon fragmentation and loss in the optic nerve of the NMO-rats compared to the Control-rat detected by NF-M immunostaining. Scale bars = 20 μm
Figure Legend Snippet: Axonal damage and loss in the spinal cord and optic nerve of the NMO-rat. a Axon injury detected in the NMO-rat compared to the Control-rat (rats infused with IgG AQP4+ 2 and IgG Control 2, D7) using neurofilament immunostaining: reduced number of axons detected as NF-M-positive spots in the white matter (WM); fragmentation and reduced axon thickness in the gray matter (GM). b Classification (10–20 to 100–140 μm 2 , ImageJ) and quantification (mean by field) of NF-M-positive spots in the spinal cord of the NMO-rats ( n = 6) compared to the Control-rats ( n = 6): loss of axons with 60–140 μm 2 sections in the NMO-rats (in cart: evaluation of the total axon number, p = 0.03). c Co-detection of myelin alteration (MBP in green ) and axonal loss (neurofilament NF-M subtype in red ) in the spinal cord of the NMO-rat compared to the Control-rat. d Increased expression of the NF-H phosphorylated form, a marker of axon injury, detected by Western blot (pNF-H/NF-H ratio; p = 0.04). e Axon fragmentation and loss in the optic nerve of the NMO-rats compared to the Control-rat detected by NF-M immunostaining. Scale bars = 20 μm

Techniques Used: Immunostaining, Expressing, Marker, Western Blot

37) Product Images from "Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes"

Article Title: Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

Journal: The EMBO Journal

doi: 10.1093/emboj/19.22.6173

Fig. 6. Vav-2 potentiates antigen receptor-induced Ca 2+ mobilization in B cells but not T cells. Jurkat T cells or Bal-17 B cells were infected with non-recombinant vaccinia (WR) or recombinant vaccinia expressing FLAG-tagged versions of Vav-1, Vav-2 or Δ1–180 Vav-2, as indicated. After infection, the cells were loaded with Indo-1. The Jurkat T cells (upper panel) were stimulated with a combination of anti-CD3 mAb (2.5 µg/ml) and goat anti-mouse IgG F(ab′) 2 . The Bal-17 B cells (middle and lower panels) were stimulated with the anti-murine IgM mAb b7.6 (5 µg/ml). The samples were immediately analyzed by flow cytometry over the indicated time course. Panel insets: protein expression in the infected cells was verified by immunoblotting of whole-cell lysates with the anti-FLAG mAb (lanes 1, 2 and 3 represent WR, Vav-1 and Vav-2 in the upper and middle panels, and WR, Vav-2 and Δ1–180 Vav-2 in the lower panel, respectively). The data shown are representative of at least three independent experiments.
Figure Legend Snippet: Fig. 6. Vav-2 potentiates antigen receptor-induced Ca 2+ mobilization in B cells but not T cells. Jurkat T cells or Bal-17 B cells were infected with non-recombinant vaccinia (WR) or recombinant vaccinia expressing FLAG-tagged versions of Vav-1, Vav-2 or Δ1–180 Vav-2, as indicated. After infection, the cells were loaded with Indo-1. The Jurkat T cells (upper panel) were stimulated with a combination of anti-CD3 mAb (2.5 µg/ml) and goat anti-mouse IgG F(ab′) 2 . The Bal-17 B cells (middle and lower panels) were stimulated with the anti-murine IgM mAb b7.6 (5 µg/ml). The samples were immediately analyzed by flow cytometry over the indicated time course. Panel insets: protein expression in the infected cells was verified by immunoblotting of whole-cell lysates with the anti-FLAG mAb (lanes 1, 2 and 3 represent WR, Vav-1 and Vav-2 in the upper and middle panels, and WR, Vav-2 and Δ1–180 Vav-2 in the lower panel, respectively). The data shown are representative of at least three independent experiments.

Techniques Used: Infection, Recombinant, Expressing, Flow Cytometry, Cytometry

Fig. 7. Ligation of CD19 results in tyrosine phosphorylation of Vav-2 and the activation of NFAT. ( A ) CD19 ligation induces tyrosine phosphorylation of Vav-2. Bal-17 B cells were incubated with the indicated doses of biotinylated Fab fragment of antibody to CD19 and then stimulated by the addition of soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 antibodies, and immunoblotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed with pooled mAbs to Vav-2. ( B ) Tyrosine phosphorylation of Vav-2 is enhanced by co-ligation of CD19 to BCR. Bal-17 B cells were incubated with the indicated doses of either control Fab, biotinylated Fab anti-CD19, or biotinylated Fab anti-κ alone or combined with 0.2 µg of biotinylated Fab anti-CD19, followed by crosslinking with soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 and immuno blotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed as above. ( C ) This experiment was performed as in (B) except that the stimulating dose of Fab anti-CD19 was 5 µg/ml. ( D ) Vav-2 potentiates CD19 induction of NFAT. Bal-17 B cells were co-transfected with p(NFAT) 3 IL-2-luc together with 1 µg of empty vector or plasmids encoding Vav-2, L212R Vav-2 or R688A Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with 1D3 anti-CD19, which was crosslinked with F(ab′) 2 goat anti-rat IgG before cell lysates were prepared and assayed for luciferase activity.
Figure Legend Snippet: Fig. 7. Ligation of CD19 results in tyrosine phosphorylation of Vav-2 and the activation of NFAT. ( A ) CD19 ligation induces tyrosine phosphorylation of Vav-2. Bal-17 B cells were incubated with the indicated doses of biotinylated Fab fragment of antibody to CD19 and then stimulated by the addition of soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 antibodies, and immunoblotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed with pooled mAbs to Vav-2. ( B ) Tyrosine phosphorylation of Vav-2 is enhanced by co-ligation of CD19 to BCR. Bal-17 B cells were incubated with the indicated doses of either control Fab, biotinylated Fab anti-CD19, or biotinylated Fab anti-κ alone or combined with 0.2 µg of biotinylated Fab anti-CD19, followed by crosslinking with soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 and immuno blotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed as above. ( C ) This experiment was performed as in (B) except that the stimulating dose of Fab anti-CD19 was 5 µg/ml. ( D ) Vav-2 potentiates CD19 induction of NFAT. Bal-17 B cells were co-transfected with p(NFAT) 3 IL-2-luc together with 1 µg of empty vector or plasmids encoding Vav-2, L212R Vav-2 or R688A Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with 1D3 anti-CD19, which was crosslinked with F(ab′) 2 goat anti-rat IgG before cell lysates were prepared and assayed for luciferase activity.

Techniques Used: Ligation, Activation Assay, Incubation, Avidin-Biotin Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay

Fig. 9. Vav-2 potentiates BCR- and CD19-driven NFAT-dependent CD5 transcription. ( A ) Overexpression of Vav-2 augments BCR- and CD19-stimulated CD5 enhancer activity. Bal-17 B cells were co-transfected with pCD5-luc122R together with 5 µg of empty vector or plasmid encoding Vav-2 or L212R Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with either b7.6 anti-IgM or 1D3 anti-CD19 with F(ab′) 2 goat anti-rat IgG and assayed for luciferase activity. ( B ) Induction of CD5 by Vav-2 requires intact NFAT regulatory sites. Bal-17 B cells were co-transfected with 10 µg of enhancerless CD5 reporter construct, the wild-type enhancer pCD5-luc122R construct or a derivative with point mutations in both the proximal and distal NFAT sites together with 5 µg of empty vector or 5 µg of plasmid encoding Vav-2. Stimulations are as in (A).
Figure Legend Snippet: Fig. 9. Vav-2 potentiates BCR- and CD19-driven NFAT-dependent CD5 transcription. ( A ) Overexpression of Vav-2 augments BCR- and CD19-stimulated CD5 enhancer activity. Bal-17 B cells were co-transfected with pCD5-luc122R together with 5 µg of empty vector or plasmid encoding Vav-2 or L212R Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with either b7.6 anti-IgM or 1D3 anti-CD19 with F(ab′) 2 goat anti-rat IgG and assayed for luciferase activity. ( B ) Induction of CD5 by Vav-2 requires intact NFAT regulatory sites. Bal-17 B cells were co-transfected with 10 µg of enhancerless CD5 reporter construct, the wild-type enhancer pCD5-luc122R construct or a derivative with point mutations in both the proximal and distal NFAT sites together with 5 µg of empty vector or 5 µg of plasmid encoding Vav-2. Stimulations are as in (A).

Techniques Used: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Cell Culture, Incubation, Luciferase, Construct

38) Product Images from "Expression of reticulon 3 in Alzheimer's disease brain"

Article Title: Expression of reticulon 3 in Alzheimer's disease brain

Journal: Neuropathology and applied neurobiology

doi: 10.1111/j.1365-2990.2008.00974.x

RTN3 protein expression in control and AD brains. (A) Membrane proteins from control and AD brains were subjected to immunoblotting with RTN3 antibody. The membrane was reprobed with β-actin antibody. (B) Band intensities in (A) were quantified
Figure Legend Snippet: RTN3 protein expression in control and AD brains. (A) Membrane proteins from control and AD brains were subjected to immunoblotting with RTN3 antibody. The membrane was reprobed with β-actin antibody. (B) Band intensities in (A) were quantified

Techniques Used: Expressing

Interactions between RTN3 and BACE1 in the brain. (A) Western blotting with the anti-RTN3 antibody (RN3-C) led to the specific detection of RTN3 proteins (~28 kDa) in mouse brain extracts. (B) Membrane extracts of mouse cerebral cortical tissues were
Figure Legend Snippet: Interactions between RTN3 and BACE1 in the brain. (A) Western blotting with the anti-RTN3 antibody (RN3-C) led to the specific detection of RTN3 proteins (~28 kDa) in mouse brain extracts. (B) Membrane extracts of mouse cerebral cortical tissues were

Techniques Used: Western Blot

Immunohistochemical analysis of RTN3 expression in the cerebral cortices of AD and control brains. (A–D) Distribution of immunoreactivity for RTN3 in the temporal cortices of AD (A, B) and control (C, D) brains. RTN3 immunoreactivity was observed
Figure Legend Snippet: Immunohistochemical analysis of RTN3 expression in the cerebral cortices of AD and control brains. (A–D) Distribution of immunoreactivity for RTN3 in the temporal cortices of AD (A, B) and control (C, D) brains. RTN3 immunoreactivity was observed

Techniques Used: Immunohistochemistry, Expressing

Analyses of the subcellular distribution of RTN3 in human cerebral cortical tissues (A) Cerebral cortical tissues from a control case were subjected to biochemical subcellular fractionation with iodixanol, followed by Western blotting with an anti-RTN3
Figure Legend Snippet: Analyses of the subcellular distribution of RTN3 in human cerebral cortical tissues (A) Cerebral cortical tissues from a control case were subjected to biochemical subcellular fractionation with iodixanol, followed by Western blotting with an anti-RTN3

Techniques Used: Fractionation, Western Blot

39) Product Images from "Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes"

Article Title: Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

Journal: The EMBO Journal

doi: 10.1093/emboj/19.22.6173

Fig. 6. Vav-2 potentiates antigen receptor-induced Ca 2+ mobilization in B cells but not T cells. Jurkat T cells or Bal-17 B cells were infected with non-recombinant vaccinia (WR) or recombinant vaccinia expressing FLAG-tagged versions of Vav-1, Vav-2 or Δ1–180 Vav-2, as indicated. After infection, the cells were loaded with Indo-1. The Jurkat T cells (upper panel) were stimulated with a combination of anti-CD3 mAb (2.5 µg/ml) and goat anti-mouse IgG F(ab′) 2 . The Bal-17 B cells (middle and lower panels) were stimulated with the anti-murine IgM mAb b7.6 (5 µg/ml). The samples were immediately analyzed by flow cytometry over the indicated time course. Panel insets: protein expression in the infected cells was verified by immunoblotting of whole-cell lysates with the anti-FLAG mAb (lanes 1, 2 and 3 represent WR, Vav-1 and Vav-2 in the upper and middle panels, and WR, Vav-2 and Δ1–180 Vav-2 in the lower panel, respectively). The data shown are representative of at least three independent experiments.
Figure Legend Snippet: Fig. 6. Vav-2 potentiates antigen receptor-induced Ca 2+ mobilization in B cells but not T cells. Jurkat T cells or Bal-17 B cells were infected with non-recombinant vaccinia (WR) or recombinant vaccinia expressing FLAG-tagged versions of Vav-1, Vav-2 or Δ1–180 Vav-2, as indicated. After infection, the cells were loaded with Indo-1. The Jurkat T cells (upper panel) were stimulated with a combination of anti-CD3 mAb (2.5 µg/ml) and goat anti-mouse IgG F(ab′) 2 . The Bal-17 B cells (middle and lower panels) were stimulated with the anti-murine IgM mAb b7.6 (5 µg/ml). The samples were immediately analyzed by flow cytometry over the indicated time course. Panel insets: protein expression in the infected cells was verified by immunoblotting of whole-cell lysates with the anti-FLAG mAb (lanes 1, 2 and 3 represent WR, Vav-1 and Vav-2 in the upper and middle panels, and WR, Vav-2 and Δ1–180 Vav-2 in the lower panel, respectively). The data shown are representative of at least three independent experiments.

Techniques Used: Infection, Recombinant, Expressing, Flow Cytometry, Cytometry

Fig. 7. Ligation of CD19 results in tyrosine phosphorylation of Vav-2 and the activation of NFAT. ( A ) CD19 ligation induces tyrosine phosphorylation of Vav-2. Bal-17 B cells were incubated with the indicated doses of biotinylated Fab fragment of antibody to CD19 and then stimulated by the addition of soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 antibodies, and immunoblotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed with pooled mAbs to Vav-2. ( B ) Tyrosine phosphorylation of Vav-2 is enhanced by co-ligation of CD19 to BCR. Bal-17 B cells were incubated with the indicated doses of either control Fab, biotinylated Fab anti-CD19, or biotinylated Fab anti-κ alone or combined with 0.2 µg of biotinylated Fab anti-CD19, followed by crosslinking with soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 and immuno blotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed as above. ( C ) This experiment was performed as in (B) except that the stimulating dose of Fab anti-CD19 was 5 µg/ml. ( D ) Vav-2 potentiates CD19 induction of NFAT. Bal-17 B cells were co-transfected with p(NFAT) 3 IL-2-luc together with 1 µg of empty vector or plasmids encoding Vav-2, L212R Vav-2 or R688A Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with 1D3 anti-CD19, which was crosslinked with F(ab′) 2 goat anti-rat IgG before cell lysates were prepared and assayed for luciferase activity.
Figure Legend Snippet: Fig. 7. Ligation of CD19 results in tyrosine phosphorylation of Vav-2 and the activation of NFAT. ( A ) CD19 ligation induces tyrosine phosphorylation of Vav-2. Bal-17 B cells were incubated with the indicated doses of biotinylated Fab fragment of antibody to CD19 and then stimulated by the addition of soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 antibodies, and immunoblotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed with pooled mAbs to Vav-2. ( B ) Tyrosine phosphorylation of Vav-2 is enhanced by co-ligation of CD19 to BCR. Bal-17 B cells were incubated with the indicated doses of either control Fab, biotinylated Fab anti-CD19, or biotinylated Fab anti-κ alone or combined with 0.2 µg of biotinylated Fab anti-CD19, followed by crosslinking with soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 and immuno blotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed as above. ( C ) This experiment was performed as in (B) except that the stimulating dose of Fab anti-CD19 was 5 µg/ml. ( D ) Vav-2 potentiates CD19 induction of NFAT. Bal-17 B cells were co-transfected with p(NFAT) 3 IL-2-luc together with 1 µg of empty vector or plasmids encoding Vav-2, L212R Vav-2 or R688A Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with 1D3 anti-CD19, which was crosslinked with F(ab′) 2 goat anti-rat IgG before cell lysates were prepared and assayed for luciferase activity.

Techniques Used: Ligation, Activation Assay, Incubation, Avidin-Biotin Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay

40) Product Images from "Deletion of OTX2 in neural ectoderm delays anterior pituitary development"

Article Title: Deletion of OTX2 in neural ectoderm delays anterior pituitary development

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddu506

(A) Loss of OTX2 in the ventral diencephalon does not affect cell specification in the anterior pituitary lobe. Immunohistochemistry at P10 on coronal sections for the hormones TSHβ, ACTH, LHβ and GH demonstrate no difference between control and the Otx2 FX ;Nkx2.1-cre mutants. Immunohistochemistry on coronal sections at P2 for AVP expression in the posterior lobe reveals no difference between control and the Otx2 FX ;Nkx2.1-cre mutants. All photos were taken at ×100. Scale bar: 100 μm. (B) The recovering, mutant infundibulum expresses Fgf10. In situ hybridization at e14.5 and e16.5 using coronal sections shows Fg10 expression in the control infundibulum (left panel). Variable expression on Fgf10 is also detected in the small infundibulum tissue of the Otx2 FX ;Nkx.1-cre mutants at e14.5 and e16.5 (arrows, middle and right panels). The photos in the top panel were taken at. Scale bar: 50 μm. The photos in the bottom panel were taken at. Scale bar: 100 μm.
Figure Legend Snippet: (A) Loss of OTX2 in the ventral diencephalon does not affect cell specification in the anterior pituitary lobe. Immunohistochemistry at P10 on coronal sections for the hormones TSHβ, ACTH, LHβ and GH demonstrate no difference between control and the Otx2 FX ;Nkx2.1-cre mutants. Immunohistochemistry on coronal sections at P2 for AVP expression in the posterior lobe reveals no difference between control and the Otx2 FX ;Nkx2.1-cre mutants. All photos were taken at ×100. Scale bar: 100 μm. (B) The recovering, mutant infundibulum expresses Fgf10. In situ hybridization at e14.5 and e16.5 using coronal sections shows Fg10 expression in the control infundibulum (left panel). Variable expression on Fgf10 is also detected in the small infundibulum tissue of the Otx2 FX ;Nkx.1-cre mutants at e14.5 and e16.5 (arrows, middle and right panels). The photos in the top panel were taken at. Scale bar: 50 μm. The photos in the bottom panel were taken at. Scale bar: 100 μm.

Techniques Used: Immunohistochemistry, Expressing, Mutagenesis, In Situ Hybridization

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    Jackson Immuno biotin conjugated goat anti rabbit igg
    Secretion of <t>ATX</t> and HECs and transfected MDCK cells. ( a ) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit <t>IgG</t> as a control. The results shown
    Biotin Conjugated Goat Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno biotin sp conjugated affinipure goat anti rabbit immunoglobulin g
    Secretion of <t>ATX</t> and HECs and transfected MDCK cells. ( a ) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit <t>IgG</t> as a control. The results shown
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    Secretion of ATX and HECs and transfected MDCK cells. ( a ) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit IgG as a control. The results shown

    Journal: Nature immunology

    Article Title: Autotaxin, a lysophosphatidic acid-producing ectoenzyme, promotes lymphocyte entry into secondary lymphoid organs

    doi: 10.1038/ni1573

    Figure Lengend Snippet: Secretion of ATX and HECs and transfected MDCK cells. ( a ) Conditioned medium from isolated HECs was subjected to SDS-PAGE and immunoblotted for ATX and GlyCAM-1 The membrane was stripped and reprobed with normal rabbit IgG as a control. The results shown

    Article Snippet: 10 µm thick cryostat sections were fixed in acetone, blocked with 3% BSA in PBS containing 10% goat serum and then stained with anti-ATX as a primary antibody followed by biotin conjugated goat anti-rabbit IgG (Jackson ImmuoResearch Laboratories) and Cy2-streptavidin (Jackson ImmuoResearch Laboratories).

    Techniques: Transfection, Isolation, SDS Page