Structured Review

GE Healthcare inflammasome stimulation
Ethanol blocks <t>inflammasome</t> activation in several cell types
Inflammasome Stimulation, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 4246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation"

Article Title: Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

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

doi: 10.4049/jimmunol.1600406

Ethanol blocks inflammasome activation in several cell types
Figure Legend Snippet: Ethanol blocks inflammasome activation in several cell types

Techniques Used: Activation Assay

Organic compounds containing hydroxyl groups inhibit NLRP3 inflammasome activation and activate protein tyrosine phosphatases
Figure Legend Snippet: Organic compounds containing hydroxyl groups inhibit NLRP3 inflammasome activation and activate protein tyrosine phosphatases

Techniques Used: Activation Assay

Ethanol rapidly inhibits IL-1β release induced by NLRP3 inflammasome agonists
Figure Legend Snippet: Ethanol rapidly inhibits IL-1β release induced by NLRP3 inflammasome agonists

Techniques Used:

2) Product Images from "Expression of orexin A and its receptor 1 in the human prostate"

Article Title: Expression of orexin A and its receptor 1 in the human prostate

Journal: Journal of Anatomy

doi: 10.1111/joa.12030

Expression of prepro-orexin and OX1R mRNAs and the proteins in human normal and hyperplastic prostates (A and B) and expression of OX1R in PNT1A cultured cells (C). (A) RT-PCR analysis. Lane 1, DNA ladder; lane 2, prepro-orexin and OX1R mRNA transcripts from whole rat brain (positive control); lanes 3 and 4, prepro-orexin and OX1R mRNA transcripts from normal and hyperplastic prostate samples, respectively; lane 5, negative control (no cDNA input). The bottom of (A) reports the beta-actin mRNA transcripts (internal control). (B) Western blotting analysis. Lane 1, homogenates from whole rat brain (positive control); lanes 2 and 3, homogenates from normal and hyperplastic prostate tissue, respectively; lane 4, prostate homogenates treated with the antisera directed against prepro-orexin or OX1R pre-absorbed with their respective control peptides (negative control). (C) Western blotting analysis. Lane 1, homogenates from whole rat brain (positive control); lane 2, homogenates from PNT1A cell lysate; lane 3, cell lysates treated with the antiserum directed against OX1R pre-absorbed with its control peptide (negative control). The upper blots of (B) and (C) were stripped and re-probed with an anti-tubulin monoclonal antibody to ensure equal loading of proteins in all lanes (lower blots). Molecular mass markers are indicated on the left of the Western blotting panels. Similar results were obtained from four separate experiments of identical design.
Figure Legend Snippet: Expression of prepro-orexin and OX1R mRNAs and the proteins in human normal and hyperplastic prostates (A and B) and expression of OX1R in PNT1A cultured cells (C). (A) RT-PCR analysis. Lane 1, DNA ladder; lane 2, prepro-orexin and OX1R mRNA transcripts from whole rat brain (positive control); lanes 3 and 4, prepro-orexin and OX1R mRNA transcripts from normal and hyperplastic prostate samples, respectively; lane 5, negative control (no cDNA input). The bottom of (A) reports the beta-actin mRNA transcripts (internal control). (B) Western blotting analysis. Lane 1, homogenates from whole rat brain (positive control); lanes 2 and 3, homogenates from normal and hyperplastic prostate tissue, respectively; lane 4, prostate homogenates treated with the antisera directed against prepro-orexin or OX1R pre-absorbed with their respective control peptides (negative control). (C) Western blotting analysis. Lane 1, homogenates from whole rat brain (positive control); lane 2, homogenates from PNT1A cell lysate; lane 3, cell lysates treated with the antiserum directed against OX1R pre-absorbed with its control peptide (negative control). The upper blots of (B) and (C) were stripped and re-probed with an anti-tubulin monoclonal antibody to ensure equal loading of proteins in all lanes (lower blots). Molecular mass markers are indicated on the left of the Western blotting panels. Similar results were obtained from four separate experiments of identical design.

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Western Blot

3) Product Images from "Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro"

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro

Journal: The American Journal of Pathology

doi:

Transdifferentiated cells exhibit increased migration in response to PDGF-BB. A: Av 17 cells cultured in growth medium ( hatched bars ) or in reduced medium ( solid bars ) for 6 days were tested for the ability to migrate toward EBM with 0.1% bovine serum albumin (control), EBM with 5% FBS (serum), 1 ng/ml bFGF, 10 ng/ml PDGF-BB, 50 ng/ml PDGF-BB, or 1 ng/ml TGF-β1. B: A neutralizing anti-PDGF-BB monoclonal antibody (mAb) and an isotype-matched IgG were tested for the ability to block the migration of av 17 cells, which were grown in reduced medium for 6 days, toward 10 ng/ml PDGF-BB. C: Av 15 cells cultured in growth medium ( hatched bars ) or in growth medium with 1 ng/ml TGF-β1 ( solid bars ) for 6 days were tested as in A . D: Checkerboard analysis was performed on av 17 cells grown in reduced medium for 6 days. PDGF-BB at 0, 1, 10, or 50 ng/ml was added to top and bottom wells as indicated, and cells allowed to migrate for 4 hours. The shaded boxes on the diagonal highlight the finding that PDGF-BB does not elicit random cell migration toward increasing concentrations of PDGF-BB in the upper and lower chambers.
Figure Legend Snippet: Transdifferentiated cells exhibit increased migration in response to PDGF-BB. A: Av 17 cells cultured in growth medium ( hatched bars ) or in reduced medium ( solid bars ) for 6 days were tested for the ability to migrate toward EBM with 0.1% bovine serum albumin (control), EBM with 5% FBS (serum), 1 ng/ml bFGF, 10 ng/ml PDGF-BB, 50 ng/ml PDGF-BB, or 1 ng/ml TGF-β1. B: A neutralizing anti-PDGF-BB monoclonal antibody (mAb) and an isotype-matched IgG were tested for the ability to block the migration of av 17 cells, which were grown in reduced medium for 6 days, toward 10 ng/ml PDGF-BB. C: Av 15 cells cultured in growth medium ( hatched bars ) or in growth medium with 1 ng/ml TGF-β1 ( solid bars ) for 6 days were tested as in A . D: Checkerboard analysis was performed on av 17 cells grown in reduced medium for 6 days. PDGF-BB at 0, 1, 10, or 50 ng/ml was added to top and bottom wells as indicated, and cells allowed to migrate for 4 hours. The shaded boxes on the diagonal highlight the finding that PDGF-BB does not elicit random cell migration toward increasing concentrations of PDGF-BB in the upper and lower chambers.

Techniques Used: Migration, Cell Culture, Blocking Assay

4) Product Images from "Spleen-Derived Interleukin-10 Downregulates the Severity of High-Fat Diet-Induced Non-Alcoholic Fatty Pancreas Disease"

Article Title: Spleen-Derived Interleukin-10 Downregulates the Severity of High-Fat Diet-Induced Non-Alcoholic Fatty Pancreas Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053154

Systemic administration of IL-10 diminishes SPX-induced fat accumulation, infiltration of macrophages, and pro-inflammatory responses in the pancreas. ( A ) Representative images of H E staining, oil-red O staining, CD11c staining, and CD206 staining in intra-lobular areas in pancreas sections from each group. Scale bar = 100 µm. ( B−E ) TG content ( B ), M1/M2 ratios ( C ), content of pro- and anti-inflammatory cytokines ( D ), and IL-10/IL-1β ratios ( E ) in the pancreas of each group ( n = 6). * P
Figure Legend Snippet: Systemic administration of IL-10 diminishes SPX-induced fat accumulation, infiltration of macrophages, and pro-inflammatory responses in the pancreas. ( A ) Representative images of H E staining, oil-red O staining, CD11c staining, and CD206 staining in intra-lobular areas in pancreas sections from each group. Scale bar = 100 µm. ( B−E ) TG content ( B ), M1/M2 ratios ( C ), content of pro- and anti-inflammatory cytokines ( D ), and IL-10/IL-1β ratios ( E ) in the pancreas of each group ( n = 6). * P

Techniques Used: Staining

SPX has little effect on the infiltration of macrophages in islets of IL-10-deficient mice. ( A and B ) Representative images of CD11c staining ( A ) and CD206 staining ( B ) in intra-islet areas of pancreas sections from each group. Scale bar = 100 µm. ( C−E ) CD11c-positive areas ( C ), CD206-positive areas ( D ), and M1/M2 ratios ( E ) in the islets in each group ( n = 6). * P
Figure Legend Snippet: SPX has little effect on the infiltration of macrophages in islets of IL-10-deficient mice. ( A and B ) Representative images of CD11c staining ( A ) and CD206 staining ( B ) in intra-islet areas of pancreas sections from each group. Scale bar = 100 µm. ( C−E ) CD11c-positive areas ( C ), CD206-positive areas ( D ), and M1/M2 ratios ( E ) in the islets in each group ( n = 6). * P

Techniques Used: Mouse Assay, Staining

Systemic administration of IL-10 diminishes SPX-induced fat accumulation, infiltration of macrophages, and pro-inflammatory responses in the islets. ( A ) Representative images of CD11c staining (upper sections) and CD206 staining (lower sections) in intra-islet areas in pancreas sections from each group. Scale bar = 100 µm. ( B−D ) CD11c-positive areas ( B ), CD206-positive areas ( C ), and M1/M2 ratios ( D ) in the islets in each group ( n = 6). * P
Figure Legend Snippet: Systemic administration of IL-10 diminishes SPX-induced fat accumulation, infiltration of macrophages, and pro-inflammatory responses in the islets. ( A ) Representative images of CD11c staining (upper sections) and CD206 staining (lower sections) in intra-islet areas in pancreas sections from each group. Scale bar = 100 µm. ( B−D ) CD11c-positive areas ( B ), CD206-positive areas ( C ), and M1/M2 ratios ( D ) in the islets in each group ( n = 6). * P

Techniques Used: Staining

SPX has little effect on the infiltration of macrophages in intra-lobular area of IL-10-deficient mice. ( A and B ) Representative images of CD11c staining ( A ) and CD206 staining ( B ) in intra-lobular areas in pancreas sections from each group. Scale bar = 100 µm. ( C−E ) CD11c-positive areas ( C ), CD206-positive areas ( D ), and M1/M2 ratios ( E ) in intra-lobular areas in each group ( n = 6). * P
Figure Legend Snippet: SPX has little effect on the infiltration of macrophages in intra-lobular area of IL-10-deficient mice. ( A and B ) Representative images of CD11c staining ( A ) and CD206 staining ( B ) in intra-lobular areas in pancreas sections from each group. Scale bar = 100 µm. ( C−E ) CD11c-positive areas ( C ), CD206-positive areas ( D ), and M1/M2 ratios ( E ) in intra-lobular areas in each group ( n = 6). * P

Techniques Used: Mouse Assay, Staining

Effects of SPX on the infiltration of M1 and M2 macrophages and on inflammatory responses in the pancreas. ( A and B ) Representative images of CD11c staining (left) and CD206 staining (right) in intra-islet areas ( A ) and intra-lobular areas ( B ) in pancreas sections from each group. ( C−E ) Percentage of CD11c-positive area ( C ) and CD206-positive area ( D ) and M1/M2 ratio ( E ) in intra-islet areas. ( F−H ) Percentage of CD11c-positive area ( F ) and CD206-positive area ( G ) and M1/M2 ratio ( H ) in intra-lobular areas. ( J and K ) Content of pro- and anti-inflammatory cytokines ( J ) and interleukin (IL)-10/IL-1β ratio ( K ) in the pancreas in each group ( n = 6). * P
Figure Legend Snippet: Effects of SPX on the infiltration of M1 and M2 macrophages and on inflammatory responses in the pancreas. ( A and B ) Representative images of CD11c staining (left) and CD206 staining (right) in intra-islet areas ( A ) and intra-lobular areas ( B ) in pancreas sections from each group. ( C−E ) Percentage of CD11c-positive area ( C ) and CD206-positive area ( D ) and M1/M2 ratio ( E ) in intra-islet areas. ( F−H ) Percentage of CD11c-positive area ( F ) and CD206-positive area ( G ) and M1/M2 ratio ( H ) in intra-lobular areas. ( J and K ) Content of pro- and anti-inflammatory cytokines ( J ) and interleukin (IL)-10/IL-1β ratio ( K ) in the pancreas in each group ( n = 6). * P

Techniques Used: Staining

5) Product Images from "Comparative interactomics analysis reveals potential regulators of α6β4 distribution in keratinocytes"

Article Title: Comparative interactomics analysis reveals potential regulators of α6β4 distribution in keratinocytes

Journal: Biology Open

doi: 10.1242/bio.054155

BioID method to identify β4 proximity interactors. (A) Schematic representation of wild-type β4 and the β4-BirA* fusion proteins. Black boxes indicate the FnIII domains. TM is transmembrane domain. (B) Western blot analysis of whole cell lysates from PA-JEB/β4 and PA-JEB/β4-BirA* keratinocytes probed with anti-β4 and anti-α-tubulin (loading control) antibodies. (C) Representative confocal microscopy images of PA-JEB keratinocytes expressing wild-type β4 and β4-BirA* stained for β4 and plectin. Scale bars: 10 μm. (D) Whole cell lysates from PA-JEB/β4-BirA* keratinocytes treated for the indicated time points with 50 μM biotin and analyzed by western blot with streptavidin-HRP. (E) Western blot analysis of biotinylated proteins from PA-JEB/β4-BirA* cells, cultured in regular medium or biotin-depleted medium for 20 h and subsequently treated with or without biotin for 24 h. Biotinylated proteins were detected by probing the membrane with Streptavidin-HRP. (F) Volcano plot showing enrichment (log 2 ) and corresponding significance ( P -value, log 10 ) of biotinylated proteins in biotin-treated and -untreated PA-JEB/β4-BirA* keratinocytes ( n =3).
Figure Legend Snippet: BioID method to identify β4 proximity interactors. (A) Schematic representation of wild-type β4 and the β4-BirA* fusion proteins. Black boxes indicate the FnIII domains. TM is transmembrane domain. (B) Western blot analysis of whole cell lysates from PA-JEB/β4 and PA-JEB/β4-BirA* keratinocytes probed with anti-β4 and anti-α-tubulin (loading control) antibodies. (C) Representative confocal microscopy images of PA-JEB keratinocytes expressing wild-type β4 and β4-BirA* stained for β4 and plectin. Scale bars: 10 μm. (D) Whole cell lysates from PA-JEB/β4-BirA* keratinocytes treated for the indicated time points with 50 μM biotin and analyzed by western blot with streptavidin-HRP. (E) Western blot analysis of biotinylated proteins from PA-JEB/β4-BirA* cells, cultured in regular medium or biotin-depleted medium for 20 h and subsequently treated with or without biotin for 24 h. Biotinylated proteins were detected by probing the membrane with Streptavidin-HRP. (F) Volcano plot showing enrichment (log 2 ) and corresponding significance ( P -value, log 10 ) of biotinylated proteins in biotin-treated and -untreated PA-JEB/β4-BirA* keratinocytes ( n =3).

Techniques Used: Western Blot, Confocal Microscopy, Expressing, Staining, Cell Culture

6) Product Images from "Pathophysiological role of microRNA-29 in pancreatic cancer stroma"

Article Title: Pathophysiological role of microRNA-29 in pancreatic cancer stroma

Journal: Scientific Reports

doi: 10.1038/srep11450

PSC and epithelial cell specific miR-29 loss of expression in human PDAC tumors. (a, c) In situ hybridization of miR-29 in PSCs (a) and epithelial cells (c) of normal control and PDAC patient tumors. FFPE pancreatic tissue sections from normal control and PDAC patients (n = 4/group) were subjected to miR-29a in situ hybridization. Representative images are presented as a single channel or merged (scale bar is 5 μm, 20X magnification). Hoechst nuclear stain (magenta), U6 (red), miR-29a (green), and GFAP-positive PSCs or CK19-positive epithelial cells (blue). (b, d) Corrected total cell fluorescence (CTCF) of miR-29a in PDAC tumors (n = 4) compared to control patients (n = 4) was calculated for each patient averaging six or more randomly selected GFAP-positive PSCs (b) or CK19-positive epithelial cells (d) using ImageJ analysis. Data represents mean + SEM. Statistics were generated using t-test, *p
Figure Legend Snippet: PSC and epithelial cell specific miR-29 loss of expression in human PDAC tumors. (a, c) In situ hybridization of miR-29 in PSCs (a) and epithelial cells (c) of normal control and PDAC patient tumors. FFPE pancreatic tissue sections from normal control and PDAC patients (n = 4/group) were subjected to miR-29a in situ hybridization. Representative images are presented as a single channel or merged (scale bar is 5 μm, 20X magnification). Hoechst nuclear stain (magenta), U6 (red), miR-29a (green), and GFAP-positive PSCs or CK19-positive epithelial cells (blue). (b, d) Corrected total cell fluorescence (CTCF) of miR-29a in PDAC tumors (n = 4) compared to control patients (n = 4) was calculated for each patient averaging six or more randomly selected GFAP-positive PSCs (b) or CK19-positive epithelial cells (d) using ImageJ analysis. Data represents mean + SEM. Statistics were generated using t-test, *p

Techniques Used: Expressing, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Staining, Fluorescence, Generated

7) Product Images from "Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A *"

Article Title: Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.169201

BM labeling of NBCe1-A cysteine-substituted constructs. A , representative data of BM labeling. HEK 293 cells expressing individual cysteine-substituted constructs were labeled with BM at room temperature. Cells were lysed, and NBCe1-A protein was immunoprecipitated, resolved on 7.5% SDS-PAGE, and transferred to PVDF membrane. Incorporated biotin was detected by HRP-streptavidin and ECL. Blots were stripped and probed with an anti-NBCe1 antibody to detect the amount of NBCe1-A protein in each sample. Pro, protein. B , summary of BM labeling of cysteine-substituted NBCe1-A constructs. The level of biotin incorporation in each sample was quantified by densitometry, and the signal was normalized to the amount of NBCe1-A protein present in the sample. In each experiment, the level of biotinylation was compared with C 1035 , whose labeling was set at 100%. Region between Val 829 and Asp 959 was labeled to a level similar to the background (NBCe1-A-5C − ) except Met 858 and therefore is not shown. Asterisks mark the constructs that were not analyzed due to lack of membrane expression. Data represent mean of three to five experiments ± S.E. ( error bars ).
Figure Legend Snippet: BM labeling of NBCe1-A cysteine-substituted constructs. A , representative data of BM labeling. HEK 293 cells expressing individual cysteine-substituted constructs were labeled with BM at room temperature. Cells were lysed, and NBCe1-A protein was immunoprecipitated, resolved on 7.5% SDS-PAGE, and transferred to PVDF membrane. Incorporated biotin was detected by HRP-streptavidin and ECL. Blots were stripped and probed with an anti-NBCe1 antibody to detect the amount of NBCe1-A protein in each sample. Pro, protein. B , summary of BM labeling of cysteine-substituted NBCe1-A constructs. The level of biotin incorporation in each sample was quantified by densitometry, and the signal was normalized to the amount of NBCe1-A protein present in the sample. In each experiment, the level of biotinylation was compared with C 1035 , whose labeling was set at 100%. Region between Val 829 and Asp 959 was labeled to a level similar to the background (NBCe1-A-5C − ) except Met 858 and therefore is not shown. Asterisks mark the constructs that were not analyzed due to lack of membrane expression. Data represent mean of three to five experiments ± S.E. ( error bars ).

Techniques Used: Labeling, Construct, Expressing, Immunoprecipitation, SDS Page

8) Product Images from "Topology of NBCe1 Protein Transmembrane Segment 1 and Structural Effect of Proximal Renal Tubular Acidosis (pRTA) S427L Mutation *"

Article Title: Topology of NBCe1 Protein Transmembrane Segment 1 and Structural Effect of Proximal Renal Tubular Acidosis (pRTA) S427L Mutation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.404533

BM labeling of AE1-substituted cysteines. A , representative data of BM labeling. HEK 293 cells expressing individual cysteine-substituted AE1 were labeled with BM in cell suspension and detected by streptavidin-HRP and ECL on Western blot. Blots were
Figure Legend Snippet: BM labeling of AE1-substituted cysteines. A , representative data of BM labeling. HEK 293 cells expressing individual cysteine-substituted AE1 were labeled with BM in cell suspension and detected by streptavidin-HRP and ECL on Western blot. Blots were

Techniques Used: Labeling, Expressing, Western Blot

9) Product Images from "Targeting of p32 in peritoneal carcinomatosis with intraperitoneal linTT1 peptide-guided pro-apoptotic nanoparticles"

Article Title: Targeting of p32 in peritoneal carcinomatosis with intraperitoneal linTT1 peptide-guided pro-apoptotic nanoparticles

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2017.06.005

Intraperitoneal linTT1-NWs have improved tumor selectivity over systemically administered NWs. (A) Representative fluorescence confocal images of linTT1-NW at 5 h after IP (first column) or IV injection (second column) demonstrates tumor homing for both routes of administration. Cryosections of tumor tissues were stained with a CD31 antibody to visualize the blood vessels. Green: NWs; Red: CD31, Blue: DAPI. Representative fields from multiple sections (n ≥ 3) prepared from at least 3 tumors are shown. Scale bars: 100 μm. (B) Biodistribution of linTT1-NWs injected IP or IV in non-target organs (liver and kidney) in mice bearing MKN-45P tumors. NWs were injected at a dose of 5 mg/kg, and tissues were collected after 5 h. Blue, DAPI; green: FAM. Scale bars: 100 pm. (C) Quantification of green fluorescence signal in tumor, liver and kidney after IP or IV injection of linTT1-FAM-NWs. Fluorescence signal intensity was quantified by ImageJ software and normalized for tissue area. Representative fields from multiple sections from tumors in three mice are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Intraperitoneal linTT1-NWs have improved tumor selectivity over systemically administered NWs. (A) Representative fluorescence confocal images of linTT1-NW at 5 h after IP (first column) or IV injection (second column) demonstrates tumor homing for both routes of administration. Cryosections of tumor tissues were stained with a CD31 antibody to visualize the blood vessels. Green: NWs; Red: CD31, Blue: DAPI. Representative fields from multiple sections (n ≥ 3) prepared from at least 3 tumors are shown. Scale bars: 100 μm. (B) Biodistribution of linTT1-NWs injected IP or IV in non-target organs (liver and kidney) in mice bearing MKN-45P tumors. NWs were injected at a dose of 5 mg/kg, and tissues were collected after 5 h. Blue, DAPI; green: FAM. Scale bars: 100 pm. (C) Quantification of green fluorescence signal in tumor, liver and kidney after IP or IV injection of linTT1-FAM-NWs. Fluorescence signal intensity was quantified by ImageJ software and normalized for tissue area. Representative fields from multiple sections from tumors in three mice are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Fluorescence, IV Injection, Staining, Injection, Mouse Assay, Software

10) Product Images from "MOSSY CELL AXON SYNAPTIC CONTACTS ON ECTOPIC GRANULE CELLS THAT ARE BORN FOLLOWING PILOCARPINE-INDUCED SEIZURES"

Article Title: MOSSY CELL AXON SYNAPTIC CONTACTS ON ECTOPIC GRANULE CELLS THAT ARE BORN FOLLOWING PILOCARPINE-INDUCED SEIZURES

Journal: Neuroscience letters

doi: 10.1016/j.neulet.2007.06.016

Hilar CGRP ImP-labeled terminals (LT) form synaptic contacts (small arrows) with EGC dendrites, defined by CaBP-ImG labeling (arrowheads). Unlabeled terminals (UT) are noted for comparison. A CGRP ImP-labeled DCV is present in panel B (large arrow). CaBP-ImG labeling is also present in axonal processes (unmarked ImG particles in A and C), reflecting axons of CaBP-labeled granule cells and EGCs in the hilus. The inset in B shows the labeled terminal tilted to 50° to better display the synapse. In C1, a CGRP ImP-labeled terminal is apposed to a CaBP-ImG labeled dendrite. In C2, two sections from C1, these processes form a synaptic contact. Scale Bar = 500 nm.
Figure Legend Snippet: Hilar CGRP ImP-labeled terminals (LT) form synaptic contacts (small arrows) with EGC dendrites, defined by CaBP-ImG labeling (arrowheads). Unlabeled terminals (UT) are noted for comparison. A CGRP ImP-labeled DCV is present in panel B (large arrow). CaBP-ImG labeling is also present in axonal processes (unmarked ImG particles in A and C), reflecting axons of CaBP-labeled granule cells and EGCs in the hilus. The inset in B shows the labeled terminal tilted to 50° to better display the synapse. In C1, a CGRP ImP-labeled terminal is apposed to a CaBP-ImG labeled dendrite. In C2, two sections from C1, these processes form a synaptic contact. Scale Bar = 500 nm.

Techniques Used: Labeling

11) Product Images from "M-Cell Surface ?1 Integrin Expression and Invasin-Mediated Targeting of Yersinia pseudotuberculosis to Mouse Peyer's Patch M Cells"

Article Title: M-Cell Surface ?1 Integrin Expression and Invasin-Mediated Targeting of Yersinia pseudotuberculosis to Mouse Peyer's Patch M Cells

Journal: Infection and Immunity

doi:

CLSM images of mouse Peyer’s patch FAE dual stained for M cells (a) and β1 integrins (b and c). (a and b) Surface views. UEA1-stained M cells express β1 integrins in their apical membranes (stained cells in panels a and b), whereas β1 integrin expression is absent from the apical membranes of enterocytes (unstained cells in panels a and b). (c) At a depth of 2 μm, both M cells and enterocytes express β1 integrins in their lateral membranes. Bar, 10 μm.
Figure Legend Snippet: CLSM images of mouse Peyer’s patch FAE dual stained for M cells (a) and β1 integrins (b and c). (a and b) Surface views. UEA1-stained M cells express β1 integrins in their apical membranes (stained cells in panels a and b), whereas β1 integrin expression is absent from the apical membranes of enterocytes (unstained cells in panels a and b). (c) At a depth of 2 μm, both M cells and enterocytes express β1 integrins in their lateral membranes. Bar, 10 μm.

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Expressing

12) Product Images from "NMDA receptor activation increases free radical production through nitric oxide and NOX2"

Article Title: NMDA receptor activation increases free radical production through nitric oxide and NOX2

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.0133-09.2009

Electron micrographs showing somatodendritic co-localization of NOX2 and nNOS ( A ) or NR1 ( B,C ) in somatosensory cortex. A: shows immunogold labeling (small arrows) for NOX2 located near endomembranes (em) and mitochondria (m) in two somata that also exhibit diffuse cytoplasmic immunoperoxidase labeling for nNOS (Nox2/nNOS-so). Within the neuropil, NOX2-immunogold (arrows) is seen near endomembranes (em) in a small dendrite (Nox2-de) without nNOS labeling. B, C: immunogold labeling (small arrows for NR1 in the cytoplasmic compartment of dendrites showing dense aggregated endomembrane (em) labeling for NOX2 (NOX2/NR1-de). These dendrites also contain several mitochondria (m) and coated vesicles (cv). Scale bars = 500nm.
Figure Legend Snippet: Electron micrographs showing somatodendritic co-localization of NOX2 and nNOS ( A ) or NR1 ( B,C ) in somatosensory cortex. A: shows immunogold labeling (small arrows) for NOX2 located near endomembranes (em) and mitochondria (m) in two somata that also exhibit diffuse cytoplasmic immunoperoxidase labeling for nNOS (Nox2/nNOS-so). Within the neuropil, NOX2-immunogold (arrows) is seen near endomembranes (em) in a small dendrite (Nox2-de) without nNOS labeling. B, C: immunogold labeling (small arrows for NR1 in the cytoplasmic compartment of dendrites showing dense aggregated endomembrane (em) labeling for NOX2 (NOX2/NR1-de). These dendrites also contain several mitochondria (m) and coated vesicles (cv). Scale bars = 500nm.

Techniques Used: Labeling

13) Product Images from "Recruitment of Tyrosine Phosphatase HCP (SHP-1) by the Killer Cell Inhibitory Receptor"

Article Title: Recruitment of Tyrosine Phosphatase HCP (SHP-1) by the Killer Cell Inhibitory Receptor

Journal: Immunity

doi:

Cross-Linking p58 with Antibody in NK Clones Induces Tyrosine Phosphorylation of a 58 kDa Protein Three NK clones expressing the GL183 determinant (SR70, SR47, and SR64) were pooled. The cells (3 × 10 6 per condition) were preincubated in the absence of primary antibody (minus), with F(ab′) 2 –GL183 (F–GL183), or intact GL183 (GL183). Following addition of F(ab′) 2 –goat anti-mouse IgG, cells were incubated for 1 min at 37 ° C and lysed. GL183 was added to the control samples (minus) after lysis. The GL183 immune complexes ( α p58) were collected with mouse anti-goat IgG and the supernatants were subjected to a second immunoprecipitation with anti-phosphotyrosine MAb 4G10 ( α ptyr). The immunoprecipitated samples were electrophoresed on the same gel and analyzed by Western blot with anti-phosphotyrosine ( α ptyr). The right panel represents a sequential probing of the membrane in the middle panel using antisera α cyt49. Molecular mass markers are indicated in kilodaltons on the left.
Figure Legend Snippet: Cross-Linking p58 with Antibody in NK Clones Induces Tyrosine Phosphorylation of a 58 kDa Protein Three NK clones expressing the GL183 determinant (SR70, SR47, and SR64) were pooled. The cells (3 × 10 6 per condition) were preincubated in the absence of primary antibody (minus), with F(ab′) 2 –GL183 (F–GL183), or intact GL183 (GL183). Following addition of F(ab′) 2 –goat anti-mouse IgG, cells were incubated for 1 min at 37 ° C and lysed. GL183 was added to the control samples (minus) after lysis. The GL183 immune complexes ( α p58) were collected with mouse anti-goat IgG and the supernatants were subjected to a second immunoprecipitation with anti-phosphotyrosine MAb 4G10 ( α ptyr). The immunoprecipitated samples were electrophoresed on the same gel and analyzed by Western blot with anti-phosphotyrosine ( α ptyr). The right panel represents a sequential probing of the membrane in the middle panel using antisera α cyt49. Molecular mass markers are indicated in kilodaltons on the left.

Techniques Used: Clone Assay, Expressing, Incubation, Lysis, Immunoprecipitation, Western Blot

14) Product Images from "Beta-synemin expression in cardiotoxin-injected rat skeletal muscle"

Article Title: Beta-synemin expression in cardiotoxin-injected rat skeletal muscle

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/1471-2474-8-40

Quantitation of β-synemin, α-dystrobrevin-1 and -2 expression following cardiotoxin injection. The mean relative protein levels were determined for β-synemin, α-dystrobrevin-1 and -2 (n = 9) at each time point by quantifying expression on western blots. The mean relative protein level of β-synemin and α-dystrobrevin-1 on days 1 and 28 were set at 0 and 100%, respectively. α-Dystrobrevin-2 expression at day 1 was not set at 0% because this protein was clearly expressed at that time. For other days, protein levels were calculated by dividing the density of the assayed protein band by the density of the band on day 28 and adjusting the data to a percentage.
Figure Legend Snippet: Quantitation of β-synemin, α-dystrobrevin-1 and -2 expression following cardiotoxin injection. The mean relative protein levels were determined for β-synemin, α-dystrobrevin-1 and -2 (n = 9) at each time point by quantifying expression on western blots. The mean relative protein level of β-synemin and α-dystrobrevin-1 on days 1 and 28 were set at 0 and 100%, respectively. α-Dystrobrevin-2 expression at day 1 was not set at 0% because this protein was clearly expressed at that time. For other days, protein levels were calculated by dividing the density of the assayed protein band by the density of the band on day 28 and adjusting the data to a percentage.

Techniques Used: Quantitation Assay, Expressing, Injection, Western Blot

Immunohistochemistry of β-synemin in rat skeletal muscle after injection of cardiotoxin. For the first three days following injection, β-synemin was not detected in transverse muscle sections. β-synemin expression increased from day 5 up to day 14 although sarcolemmal expression appeared to be more prominent on days 21 and 28. Panels are labeled with regards to the number of days (d) following cardiotoxin injection. Bar = 50 μm
Figure Legend Snippet: Immunohistochemistry of β-synemin in rat skeletal muscle after injection of cardiotoxin. For the first three days following injection, β-synemin was not detected in transverse muscle sections. β-synemin expression increased from day 5 up to day 14 although sarcolemmal expression appeared to be more prominent on days 21 and 28. Panels are labeled with regards to the number of days (d) following cardiotoxin injection. Bar = 50 μm

Techniques Used: Immunohistochemistry, Injection, Expressing, Labeling

Immunohistochemistry of β-synemin and ATPase staining on muscle sections after injection with cardiotoxin. (A) 10 μm transverse muscle sections were immunostained with an antibody against β-synemin. (B-D) ATPase staining on serial sections was performed at pH 4.3 (B), pH 4.6 (C), and pH 10.4 (D). Type 1 fibers (labeled with a
Figure Legend Snippet: Immunohistochemistry of β-synemin and ATPase staining on muscle sections after injection with cardiotoxin. (A) 10 μm transverse muscle sections were immunostained with an antibody against β-synemin. (B-D) ATPase staining on serial sections was performed at pH 4.3 (B), pH 4.6 (C), and pH 10.4 (D). Type 1 fibers (labeled with a "1") show strong staining at pH 4.3 and pH 4.6 and no staining at pH 10.4, whereas type 2A and 2B fibers (labeled with a "2A" and "2B") show the reverse. Type 2A fibers show weaker staining than type 2B at pH 4.3 and pH 4.6. In addition, type 2C fibers reacted at pH 4.3, pH 4.6, and pH 10.4 and are under the process of regeneration. Bar = 50 μm

Techniques Used: Immunohistochemistry, Staining, Injection, Labeling

Immunoblot analysis for β-synemin, α-dystrobrevin-1 and -2 following the injection of cardiotoxin. β-Synemin and α-dystrobrevin-1 were detected after day 7, whereas α-dystrobrevin-2 was observed as early as day 1. To normalize for expression levels, immunoblots for β-synemin, α-dystrobrevin-1 and -2 are shown from representative, but different experiments. Approximate molecular weights are indicated on the right.
Figure Legend Snippet: Immunoblot analysis for β-synemin, α-dystrobrevin-1 and -2 following the injection of cardiotoxin. β-Synemin and α-dystrobrevin-1 were detected after day 7, whereas α-dystrobrevin-2 was observed as early as day 1. To normalize for expression levels, immunoblots for β-synemin, α-dystrobrevin-1 and -2 are shown from representative, but different experiments. Approximate molecular weights are indicated on the right.

Techniques Used: Injection, Expressing, Western Blot

β-synemin, α-dystrobrevin-1 and -2 are predominantly expressed after birth. Lower-limb muscles from rats at embryonic day 16 through 7 days post-birth were harvested and analyzed using an immunoblot assay. β-synemin and α-dystrobrevin-1 were first detected 5 days after birth, while α-dystrobrevin-2 was expressed in day 20 embryos.
Figure Legend Snippet: β-synemin, α-dystrobrevin-1 and -2 are predominantly expressed after birth. Lower-limb muscles from rats at embryonic day 16 through 7 days post-birth were harvested and analyzed using an immunoblot assay. β-synemin and α-dystrobrevin-1 were first detected 5 days after birth, while α-dystrobrevin-2 was expressed in day 20 embryos.

Techniques Used:

β-synemin and α-dystrobrevin interact in vivo and co-localize in rat muscle. (A) Using an anti-α-dystrobrevin antibody, α-dystrobrevin and its interacting proteins were immunoprecipitated from rat protein muscle extracts. Western blot analysis was performed to localize the β-synemin protein. β-synemin was found to be present in the muscle extract (1) and immunoprecipitated pellet (2), while it was not detected in the supernatant (3). (B) Using an anti-β-synemin antibody, β-synemin and its interacting proteins were immunoprecipitated from rat protein muscle extracts. Western blot analysis was performed to localize the α-dystrobrevin isoforms. Both α-dystrobrevin-1 and -2 were detected in the muscle extract (1) and pellet (2), while only α-dystrobrevin-2 was prese nt in the supernatant (3). (C) Rat tibialis anterior muscle was sectioned 21 days following cardiotoxin injection and double immunostained with antibodies against α-dystrobrevin antibody (left panel), β-synemin (center panel), and the merged image (right panel). Bar = 50 μm
Figure Legend Snippet: β-synemin and α-dystrobrevin interact in vivo and co-localize in rat muscle. (A) Using an anti-α-dystrobrevin antibody, α-dystrobrevin and its interacting proteins were immunoprecipitated from rat protein muscle extracts. Western blot analysis was performed to localize the β-synemin protein. β-synemin was found to be present in the muscle extract (1) and immunoprecipitated pellet (2), while it was not detected in the supernatant (3). (B) Using an anti-β-synemin antibody, β-synemin and its interacting proteins were immunoprecipitated from rat protein muscle extracts. Western blot analysis was performed to localize the α-dystrobrevin isoforms. Both α-dystrobrevin-1 and -2 were detected in the muscle extract (1) and pellet (2), while only α-dystrobrevin-2 was prese nt in the supernatant (3). (C) Rat tibialis anterior muscle was sectioned 21 days following cardiotoxin injection and double immunostained with antibodies against α-dystrobrevin antibody (left panel), β-synemin (center panel), and the merged image (right panel). Bar = 50 μm

Techniques Used: In Vivo, Immunoprecipitation, Western Blot, Injection

15) Product Images from "Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography *"

Article Title: Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.619320

BM labeling of NBCe1-A cysteine-reintroduced constructs and detection of intramolecular disulfide bonds in NBCe1-A. A , representative data of BM labeling. HEK 293 cells expressing individual cysteine-reintroduced constructs were labeled with BM at room temperature. Cells were lysed, and NBCe1-A protein was immunoprecipitated, resolved by 7.5% SDS-PAGE, and transferred to a PVDF membrane. Incorporated biotin was detected by HRP-streptavidin and ECL. Blots were stripped and probed with an anti-NBCe1 antibody to detect the amount of NBCe1-A protein in each sample. Pro , protein. B , the β-ME-treated and untreated NBCe1-A-5C − and NBCe1-A-9C − protein samples were resolved by 7.5% SDS-PAGE, transferred to a PVDF membrane, and probed with an anti-NBCe1-A antibody. −, without β-ME; +, with β-ME.
Figure Legend Snippet: BM labeling of NBCe1-A cysteine-reintroduced constructs and detection of intramolecular disulfide bonds in NBCe1-A. A , representative data of BM labeling. HEK 293 cells expressing individual cysteine-reintroduced constructs were labeled with BM at room temperature. Cells were lysed, and NBCe1-A protein was immunoprecipitated, resolved by 7.5% SDS-PAGE, and transferred to a PVDF membrane. Incorporated biotin was detected by HRP-streptavidin and ECL. Blots were stripped and probed with an anti-NBCe1 antibody to detect the amount of NBCe1-A protein in each sample. Pro , protein. B , the β-ME-treated and untreated NBCe1-A-5C − and NBCe1-A-9C − protein samples were resolved by 7.5% SDS-PAGE, transferred to a PVDF membrane, and probed with an anti-NBCe1-A antibody. −, without β-ME; +, with β-ME.

Techniques Used: Labeling, Construct, Expressing, Immunoprecipitation, SDS Page

16) Product Images from "JAM4, a Junctional Cell Adhesion Molecule Interacting with a Tight Junction Protein, MAGI-1"

Article Title: JAM4, a Junctional Cell Adhesion Molecule Interacting with a Tight Junction Protein, MAGI-1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.12.4267-4282.2003

Cell aggregation activity of JAM4. (A) Cell aggregation assays were performed using L cells or stable transformants of L cells expressing JAM4 (L-JAM4) or JAM4ΔC (L-JAM4ΔC). The extent of cell aggregation is represented by the ratio of the total particle number at the indicated time point (N t ) to the initial particle number (N 0 ). The N t /N 0 values are representative of the means ± standard errors of three independent experiments. (B) Expression of JAM4 and JAM4ΔC. Cell surface proteins were biotinylated, precipitated by avidin-agarose beads, and then immunoblotted with the anti-JAM4 antibody. Lane 1, wild-type L cells; lane 2, L-JAM4 cells; lane 3, L-JAM4ΔC cells. (C) Cell aggregation of L-JAM4 cells. Results for wild-type L cells (L-w.t.), L-JAM4 cells (L-JAM4), and L-JAM4ΔC cells (L-JAM4ΔC) at 60 min in a cell aggregation assay are shown. Bar, 200 μm.
Figure Legend Snippet: Cell aggregation activity of JAM4. (A) Cell aggregation assays were performed using L cells or stable transformants of L cells expressing JAM4 (L-JAM4) or JAM4ΔC (L-JAM4ΔC). The extent of cell aggregation is represented by the ratio of the total particle number at the indicated time point (N t ) to the initial particle number (N 0 ). The N t /N 0 values are representative of the means ± standard errors of three independent experiments. (B) Expression of JAM4 and JAM4ΔC. Cell surface proteins were biotinylated, precipitated by avidin-agarose beads, and then immunoblotted with the anti-JAM4 antibody. Lane 1, wild-type L cells; lane 2, L-JAM4 cells; lane 3, L-JAM4ΔC cells. (C) Cell aggregation of L-JAM4 cells. Results for wild-type L cells (L-w.t.), L-JAM4 cells (L-JAM4), and L-JAM4ΔC cells (L-JAM4ΔC) at 60 min in a cell aggregation assay are shown. Bar, 200 μm.

Techniques Used: Activity Assay, Expressing, Avidin-Biotin Assay

Interaction of JAM4 with MAGI-1. (A) Coimmunoprecipitation of JAM4 and MAGI-1. Myc-MAGI-1 with either FLAG-JAM4 or FLAG-JAM4ΔC was coexpressed in COS-7 cells. Anti-MAGI-1 antibody coprecipitated FLAG-JAM4 but not FLAG-JAM4ΔC with Myc-MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. Input, cell lysates; PIS IP, immunoprecipitate with the preimmune serum; MAGI IP, immunoprecipitate with the anti-MAGI-1 serum. (B) GST pulldown assay (using GST fusion proteins containing various PDZ domains of MAGI-1) of FLAG-JAM4. Lysates of COS cells expressing FLAG-JAM4 were incubated with GST alone or GST-MAGI-1-PDZ1, -PDZ2, -PDZ3, -PDZ4, or -PDZ5 immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed by immunoblotting with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (C) Specificity of the interaction of MAGI-1 with JAM4. Myc-MAGI-1 was coexpressed with either FLAG-JAM4 or FLAG-JAM1 in COS-7 cells. Lysates were incubated with either preimmune serum or anti-MAGI-1 serum immobilized on protein G-Sepharose beads. The resulting complexes were analyzed with either the anti-Myc or anti-FLAG antibody. MAGI-1 did not bind JAM1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (D) Specificity of the interaction of JAM4 with MAGI-1. Lysates of COS-7 cells expressing Myc-MAGI-1, Myc-ZO-1-1, Myc-CASK, or Myc-Lin-7 were incubated with GST alone or GST-JAM4-C immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed by immunoblotting with the anti-Myc antibody. JAM4 did not bind ZO-1, CASK, or Lin-7. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (E) Interaction of JAM4 and MAGI-1 in COS-7 cells. Subcellular localization of Myc-MAGI-1 (a), FLAG-JAM4 (b), and FLAG-JAM4ΔC (c) in COS-7 cells is shown. (d) Myc-MAGI-1 and FLAG-JAM4 were colocalized and formed clusters in COS-7 cells when coexpressed. (e) Myc-MAGI-1 and FLAG-JAM4ΔC did not interact with each other in COS-7 cells. (f) The effect of latrunculin A on the clustering of FLAG-JAM4 and Myc-MAGI-1. COS-7 cells were transfected with pFLAG JAM4 and pClneo Myc MAGI-1 and were treated (before fixation) with 5 μM latrunculin A for 5 min.
Figure Legend Snippet: Interaction of JAM4 with MAGI-1. (A) Coimmunoprecipitation of JAM4 and MAGI-1. Myc-MAGI-1 with either FLAG-JAM4 or FLAG-JAM4ΔC was coexpressed in COS-7 cells. Anti-MAGI-1 antibody coprecipitated FLAG-JAM4 but not FLAG-JAM4ΔC with Myc-MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. Input, cell lysates; PIS IP, immunoprecipitate with the preimmune serum; MAGI IP, immunoprecipitate with the anti-MAGI-1 serum. (B) GST pulldown assay (using GST fusion proteins containing various PDZ domains of MAGI-1) of FLAG-JAM4. Lysates of COS cells expressing FLAG-JAM4 were incubated with GST alone or GST-MAGI-1-PDZ1, -PDZ2, -PDZ3, -PDZ4, or -PDZ5 immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed by immunoblotting with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (C) Specificity of the interaction of MAGI-1 with JAM4. Myc-MAGI-1 was coexpressed with either FLAG-JAM4 or FLAG-JAM1 in COS-7 cells. Lysates were incubated with either preimmune serum or anti-MAGI-1 serum immobilized on protein G-Sepharose beads. The resulting complexes were analyzed with either the anti-Myc or anti-FLAG antibody. MAGI-1 did not bind JAM1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (D) Specificity of the interaction of JAM4 with MAGI-1. Lysates of COS-7 cells expressing Myc-MAGI-1, Myc-ZO-1-1, Myc-CASK, or Myc-Lin-7 were incubated with GST alone or GST-JAM4-C immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed by immunoblotting with the anti-Myc antibody. JAM4 did not bind ZO-1, CASK, or Lin-7. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (E) Interaction of JAM4 and MAGI-1 in COS-7 cells. Subcellular localization of Myc-MAGI-1 (a), FLAG-JAM4 (b), and FLAG-JAM4ΔC (c) in COS-7 cells is shown. (d) Myc-MAGI-1 and FLAG-JAM4 were colocalized and formed clusters in COS-7 cells when coexpressed. (e) Myc-MAGI-1 and FLAG-JAM4ΔC did not interact with each other in COS-7 cells. (f) The effect of latrunculin A on the clustering of FLAG-JAM4 and Myc-MAGI-1. COS-7 cells were transfected with pFLAG JAM4 and pClneo Myc MAGI-1 and were treated (before fixation) with 5 μM latrunculin A for 5 min.

Techniques Used: GST Pulldown Assay, Expressing, Incubation, Transfection

(A) Northern blot analysis of JAM4. A uniformly labeled probe corresponding to the full length of JAM4 was prepared. Blots with 20 μg of total RNA from each mouse tissue were hybridized with the probe (3,600,000 cpm) and exposed for 8 days. The mobilities of molecular mass standards (in kilobases) are indicated at the left. (B) Western blot with the anti-JAM4 antibody of rat kidney, intestine, spleen, and MTD-1A cells. Homogenates (total protein, 20 μg) from rat kidney, small intestine, spleen, and MTD-1A cells were resolved by SDS-PAGE and immunoblotted with the affinity-purified antibody against JAM4. Lane 1, spleen cells; lanes 2 and 4, kidney cells; lanes 3 and 5, small intestine cells; lanes 6 and 7, MTD-1A cells. For lanes 4, 5, and 7, the antibody was preincubated with 5 μM immunogen. The mobilities of molecular mass standards (in kilodaltons) are indicated at left. (C) FLAG-JAM4 in various cells. (a) Homogenates (total protein, 20 μg) from various stable transformants expressing FLAG-JAM4 were resolved by SDS-PAGE and immunoblotted with the anti-FLAG antibody. (b) Homogenates of stable transformants were treated with N -glycosidase F and charged onto SDS-PAGE gel with the in vitro transcription and translation product. The lane for the in vitro transcription and translation product was separately analyzed with the image analyzer. The remaining lanes were immunoblotted with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.
Figure Legend Snippet: (A) Northern blot analysis of JAM4. A uniformly labeled probe corresponding to the full length of JAM4 was prepared. Blots with 20 μg of total RNA from each mouse tissue were hybridized with the probe (3,600,000 cpm) and exposed for 8 days. The mobilities of molecular mass standards (in kilobases) are indicated at the left. (B) Western blot with the anti-JAM4 antibody of rat kidney, intestine, spleen, and MTD-1A cells. Homogenates (total protein, 20 μg) from rat kidney, small intestine, spleen, and MTD-1A cells were resolved by SDS-PAGE and immunoblotted with the affinity-purified antibody against JAM4. Lane 1, spleen cells; lanes 2 and 4, kidney cells; lanes 3 and 5, small intestine cells; lanes 6 and 7, MTD-1A cells. For lanes 4, 5, and 7, the antibody was preincubated with 5 μM immunogen. The mobilities of molecular mass standards (in kilodaltons) are indicated at left. (C) FLAG-JAM4 in various cells. (a) Homogenates (total protein, 20 μg) from various stable transformants expressing FLAG-JAM4 were resolved by SDS-PAGE and immunoblotted with the anti-FLAG antibody. (b) Homogenates of stable transformants were treated with N -glycosidase F and charged onto SDS-PAGE gel with the in vitro transcription and translation product. The lane for the in vitro transcription and translation product was separately analyzed with the image analyzer. The remaining lanes were immunoblotted with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.

Techniques Used: Northern Blot, Labeling, Western Blot, SDS Page, Affinity Purification, Expressing, In Vitro

Effect of MAGI-1 on the cell adhesive activity of JAM4. (A) Phase-contrast images of L-FLAG-JAM4 (a), L-FLAG-JAM4ΔC (b), L-FLAG-JAM4/Myc-MAGI-1 (c), and L-FLAG-JAM4ΔC/Myc-MAGI-1 (d) cells grown in cultures on plastic dishes. Bar, 50 μm. (B) Immunofluorescence of FLAG-JAM4 in L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc-MAGI-1 (b) cells. Bar, 10 μm. (C) Phase-contrast images of L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc-MAGI-1 (b) cells in collagen cultures. Bar, 100 μm. (D) Dissociation assay of L-FLAG-JAM4 and L-FLAG-JAM4/Myc-MAGI-1 cells. (a) L-FLAG-JAM4 cells after being pipetted five times; (b) L-FLAG-JAM4/Myc-MAGI-1 cells after being pipetted five times. Bar, 200 μm. (c) Western blot with the anti-FLAG antibody of L-FLAG-JAM4 and L-FLAG-JAM4/Myc-MAGI-1 cells. Lane 1, L-FLAG-JAM4 cells; lane 2, L-FLAG-JAM4/Myc-MAGI-1 cells. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.
Figure Legend Snippet: Effect of MAGI-1 on the cell adhesive activity of JAM4. (A) Phase-contrast images of L-FLAG-JAM4 (a), L-FLAG-JAM4ΔC (b), L-FLAG-JAM4/Myc-MAGI-1 (c), and L-FLAG-JAM4ΔC/Myc-MAGI-1 (d) cells grown in cultures on plastic dishes. Bar, 50 μm. (B) Immunofluorescence of FLAG-JAM4 in L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc-MAGI-1 (b) cells. Bar, 10 μm. (C) Phase-contrast images of L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc-MAGI-1 (b) cells in collagen cultures. Bar, 100 μm. (D) Dissociation assay of L-FLAG-JAM4 and L-FLAG-JAM4/Myc-MAGI-1 cells. (a) L-FLAG-JAM4 cells after being pipetted five times; (b) L-FLAG-JAM4/Myc-MAGI-1 cells after being pipetted five times. Bar, 200 μm. (c) Western blot with the anti-FLAG antibody of L-FLAG-JAM4 and L-FLAG-JAM4/Myc-MAGI-1 cells. Lane 1, L-FLAG-JAM4 cells; lane 2, L-FLAG-JAM4/Myc-MAGI-1 cells. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.

Techniques Used: Activity Assay, Immunofluorescence, Western Blot

Effect of disruption of the cytoskeleton on JAM4/MAGI-1-mediated cell adhesion. (A) Subcellular fractionation of L-Myc-MAGI-1, L-FLAG-JAM4, and L-FLAG-JAM4/Myc-MAGI-1 cells. Comparable amounts of the fractions from various L cells were immunoblotted with the anti-Myc or the anti-FLAG antibody. Insol., insoluble. (a) L-Myc-MAGI-1 cells; (b) L-FLAG-JAM4 cells; (c) L-FLAG-JAM4/Myc-MAGI-1 cells. (B) Dissociation assay of L-FLAG-JAM4/Myc-MAGI-1 cells after mock, nocodazole, and latrunculin A treatment. L-FLAG-JAM4/Myc-MAGI-1 cells were treated with DMSO (a), 1 μg of nocodazole/ml (b), or 5 μM latrunculin A (c) for 60 min before the dissociation assay. Upper photos, cells after the treatment and before scraping; lower photos, cells after being pipetted five times. Bar, 200 μm.
Figure Legend Snippet: Effect of disruption of the cytoskeleton on JAM4/MAGI-1-mediated cell adhesion. (A) Subcellular fractionation of L-Myc-MAGI-1, L-FLAG-JAM4, and L-FLAG-JAM4/Myc-MAGI-1 cells. Comparable amounts of the fractions from various L cells were immunoblotted with the anti-Myc or the anti-FLAG antibody. Insol., insoluble. (a) L-Myc-MAGI-1 cells; (b) L-FLAG-JAM4 cells; (c) L-FLAG-JAM4/Myc-MAGI-1 cells. (B) Dissociation assay of L-FLAG-JAM4/Myc-MAGI-1 cells after mock, nocodazole, and latrunculin A treatment. L-FLAG-JAM4/Myc-MAGI-1 cells were treated with DMSO (a), 1 μg of nocodazole/ml (b), or 5 μM latrunculin A (c) for 60 min before the dissociation assay. Upper photos, cells after the treatment and before scraping; lower photos, cells after being pipetted five times. Bar, 200 μm.

Techniques Used: Fractionation

Effect of JAM4 and MAGI-1 on the permeability of CHO cell monolayers. (A) Wild-type CHO and CHO-FLAG-JAM4 cells were grown in cultures in Transwells. FITC-dextran was added to the upper chamber, and the permeability was measured. The permeability of wild-type CHO cells was taken as 100%. Data are indicated as the means ± standard errors of three independent experiments. (B) (a) Wild-type CHO and CHO-FLAG-JAM4 cells were infected by adenovirus to express either GFP or GFP-MAGI-1. At 2 h after the infection, these cells were plated on Transwells for the permeability assay. (b) Immunoblots (IB) of CHO and CHO-FLAG-JAM4 cells to confirm the protein expression. Left panel, immunoblot with the anti-GFP antibody; right panel, immunoblot with the anti-FLAG antibody. Lanes 1, wild-type CHO cells with GFP; lanes 2, wild-type CHO cells with GFP-MAGI-1; lanes 3, CHO-FLAG-JAM4 cells with GFP; lanes 4, CHO-FLAG-JAM4 cells with GFP-MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.
Figure Legend Snippet: Effect of JAM4 and MAGI-1 on the permeability of CHO cell monolayers. (A) Wild-type CHO and CHO-FLAG-JAM4 cells were grown in cultures in Transwells. FITC-dextran was added to the upper chamber, and the permeability was measured. The permeability of wild-type CHO cells was taken as 100%. Data are indicated as the means ± standard errors of three independent experiments. (B) (a) Wild-type CHO and CHO-FLAG-JAM4 cells were infected by adenovirus to express either GFP or GFP-MAGI-1. At 2 h after the infection, these cells were plated on Transwells for the permeability assay. (b) Immunoblots (IB) of CHO and CHO-FLAG-JAM4 cells to confirm the protein expression. Left panel, immunoblot with the anti-GFP antibody; right panel, immunoblot with the anti-FLAG antibody. Lanes 1, wild-type CHO cells with GFP; lanes 2, wild-type CHO cells with GFP-MAGI-1; lanes 3, CHO-FLAG-JAM4 cells with GFP; lanes 4, CHO-FLAG-JAM4 cells with GFP-MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.

Techniques Used: Permeability, Infection, Western Blot, Expressing

Immunohistological analysis of JAM4. (A) JAM4 in kidney cells. The sections of rat kidney were stained with the affinity-purified antibody in the absence (a) or the presence (b) of 5 μM immunogen. Signals were detected in glomeruli (arrow) and on apical membranes of proximal tubules (arrowheads). (a2) The demarcated area shown in panel a1 at higher magnification. Bars, 100 μm (a1 and b) and 20 μm (a2). (B) Colocalization of JAM4 and MAGI-1 with ZO-1 in kidney glomeruli. Sections of rat kidney were double stained with various antibodies. (a) JAM4 and ZO-1; (b) MAGI-1 and anti-ZO-1. Bar, 20 μm. (C) Electron microscopy localization of JAM4 in podocytes. (a) JAM4; (b) MAGI-1. Bar, 0.1 μm. (D) Colocalization of JAM4 with ZO-1 in small intestine epithelial cells. (a) JAM4 and ZO-1; (b) JAM4 and β-catenin. Bar, 50 μm.
Figure Legend Snippet: Immunohistological analysis of JAM4. (A) JAM4 in kidney cells. The sections of rat kidney were stained with the affinity-purified antibody in the absence (a) or the presence (b) of 5 μM immunogen. Signals were detected in glomeruli (arrow) and on apical membranes of proximal tubules (arrowheads). (a2) The demarcated area shown in panel a1 at higher magnification. Bars, 100 μm (a1 and b) and 20 μm (a2). (B) Colocalization of JAM4 and MAGI-1 with ZO-1 in kidney glomeruli. Sections of rat kidney were double stained with various antibodies. (a) JAM4 and ZO-1; (b) MAGI-1 and anti-ZO-1. Bar, 20 μm. (C) Electron microscopy localization of JAM4 in podocytes. (a) JAM4; (b) MAGI-1. Bar, 0.1 μm. (D) Colocalization of JAM4 with ZO-1 in small intestine epithelial cells. (a) JAM4 and ZO-1; (b) JAM4 and β-catenin. Bar, 50 μm.

Techniques Used: Staining, Affinity Purification, Electron Microscopy

Immunofluorescence analysis of JAM4 in MTD-1A and MDCK cells. (A) MTD-1A cells were fixed and immunostained with anti-JAM4, anti-MAGI-1, anti-β-catenin, and anti-ZO-1 antibodies. JAM4 and MAGI-1 were detected by anti-rabbit antibodies. β-Catenin and ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) JAM4, β-catenin, and ZO-1 in MTD-1A cells; (b) MAGI-1, β-catenin, and ZO-1 in MTD-1A cells. Bar, 20 μm. (B) MDCK-FLAG-JAM4 cells were fixed and immunostained with anti-FLAG, anti-MAGI-1, anti-ERBIN, and anti-ZO-1 antibodies. MAGI-1 and ERBIN were detected by anti-rabbit antibodies. FLAG and ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) FLAG-JAM4, MAGI-1, and ZO-1 in MDCK cells; (b) FLAG-JAM4, ERBIN, and ZO-1 in MDCK cells. Bar, 20 μm.
Figure Legend Snippet: Immunofluorescence analysis of JAM4 in MTD-1A and MDCK cells. (A) MTD-1A cells were fixed and immunostained with anti-JAM4, anti-MAGI-1, anti-β-catenin, and anti-ZO-1 antibodies. JAM4 and MAGI-1 were detected by anti-rabbit antibodies. β-Catenin and ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) JAM4, β-catenin, and ZO-1 in MTD-1A cells; (b) MAGI-1, β-catenin, and ZO-1 in MTD-1A cells. Bar, 20 μm. (B) MDCK-FLAG-JAM4 cells were fixed and immunostained with anti-FLAG, anti-MAGI-1, anti-ERBIN, and anti-ZO-1 antibodies. MAGI-1 and ERBIN were detected by anti-rabbit antibodies. FLAG and ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) FLAG-JAM4, MAGI-1, and ZO-1 in MDCK cells; (b) FLAG-JAM4, ERBIN, and ZO-1 in MDCK cells. Bar, 20 μm.

Techniques Used: Immunofluorescence

Recruitment of Myc-MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. (A) Stable transformants of L cells expressing FLAG-JAM4 (L-FLAG-JAM4) (a) and FLAG-JAM4ΔC (L-FLAG-JAM4ΔC) (b) were grown in cultures to generate cell contacts. FLAG-JAM4 and FLAG-JAM4ΔC were accumulated at cell contacts (arrows). (c) L-Myc-MAGI-1 cells. Myc-MAGI-1 was diffusely distributed. (B) Myc-MAGI-1 in L-FLAG-JAM4/Myc-MAGI-1 (a) and L-FLAG-JAM4ΔC/Myc-MAGI-1 (b) cells. FLAG-JAM4 (arrows) and Myc-MAGI-1 (arrowheads) were accumulated at cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. (C) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG and anti-ZO-1 antibodies. (b) L-FLAG-JAM4/Myc-MAGI-1 cells; (c) L-FLAG-JAM4ΔC/Myc-MAGI-1 cells. L-FLAG-JAM4/Myc-MAGI-1 cells and L-FLAG-JAM4ΔC/Myc-MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-ZO-1 antibodies. ZO-1 (arrowheads) was recruited with Myc-MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. (D) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG and anti-occludin antibodies. (b) L-FLAG-JAM4/Myc-MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-occludin antibodies. Occludin (arrowheads) was recruited with Myc-MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. Bar, 20 μm.
Figure Legend Snippet: Recruitment of Myc-MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. (A) Stable transformants of L cells expressing FLAG-JAM4 (L-FLAG-JAM4) (a) and FLAG-JAM4ΔC (L-FLAG-JAM4ΔC) (b) were grown in cultures to generate cell contacts. FLAG-JAM4 and FLAG-JAM4ΔC were accumulated at cell contacts (arrows). (c) L-Myc-MAGI-1 cells. Myc-MAGI-1 was diffusely distributed. (B) Myc-MAGI-1 in L-FLAG-JAM4/Myc-MAGI-1 (a) and L-FLAG-JAM4ΔC/Myc-MAGI-1 (b) cells. FLAG-JAM4 (arrows) and Myc-MAGI-1 (arrowheads) were accumulated at cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. (C) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG and anti-ZO-1 antibodies. (b) L-FLAG-JAM4/Myc-MAGI-1 cells; (c) L-FLAG-JAM4ΔC/Myc-MAGI-1 cells. L-FLAG-JAM4/Myc-MAGI-1 cells and L-FLAG-JAM4ΔC/Myc-MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-ZO-1 antibodies. ZO-1 (arrowheads) was recruited with Myc-MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. (D) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG and anti-occludin antibodies. (b) L-FLAG-JAM4/Myc-MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-occludin antibodies. Occludin (arrowheads) was recruited with Myc-MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. Bar, 20 μm.

Techniques Used: Expressing, Staining

17) Product Images from "Immunocytochemical Colocalization of Specific Immunoglobulin A with Sendai Virus Protein in Infected Polarized Epithelium "

Article Title: Immunocytochemical Colocalization of Specific Immunoglobulin A with Sendai Virus Protein in Infected Polarized Epithelium

Journal: The Journal of Experimental Medicine

doi:

Colocalization of specific IgA and viral protein within the cytoplasm of Sendai virus–infected cells. In polarized MDCK cells infected with Sendai virus and treated by basolateral application of IgA anti-HN 4 h later ( A , a replicate culture of Fig. 1 A ), there is colocalization of numerous large (15 nm) gold particles labeling IgA ( large arrows ) and numerous small (5 nm) gold particles labeling viral protein ( small arrows ) to form multilamellar membrane structures, located deep within the cytosol. In infected cells treated with specific IgG ( B ) or irrelevant IgA ( C ), budding virions arising from the cell surface (indicated by asterisks) are identified by anti-viral HN protein staining (15-nm gold particles in B , large arrow ; 5-nm gold particles in C , small arrow ). The intracellular inclusions which develop in infected cells treated with specific IgA are only rarely identified in cells treated with specific IgG, and Ig does not frequently colocalize with viral protein. Neither infected cells treated with irrelevant IgA nor uninfected cells ever contain these structures. Bar = 0.25 μm.
Figure Legend Snippet: Colocalization of specific IgA and viral protein within the cytoplasm of Sendai virus–infected cells. In polarized MDCK cells infected with Sendai virus and treated by basolateral application of IgA anti-HN 4 h later ( A , a replicate culture of Fig. 1 A ), there is colocalization of numerous large (15 nm) gold particles labeling IgA ( large arrows ) and numerous small (5 nm) gold particles labeling viral protein ( small arrows ) to form multilamellar membrane structures, located deep within the cytosol. In infected cells treated with specific IgG ( B ) or irrelevant IgA ( C ), budding virions arising from the cell surface (indicated by asterisks) are identified by anti-viral HN protein staining (15-nm gold particles in B , large arrow ; 5-nm gold particles in C , small arrow ). The intracellular inclusions which develop in infected cells treated with specific IgA are only rarely identified in cells treated with specific IgG, and Ig does not frequently colocalize with viral protein. Neither infected cells treated with irrelevant IgA nor uninfected cells ever contain these structures. Bar = 0.25 μm.

Techniques Used: Infection, Labeling, Staining

Treatment with specific IgA, but not specific IgG or irrelevant IgA, reduces the appearance of viral protein on the cell surface. Polarized monolayers of MDCK cells in culture well inserts, stably transfected with the pIgR derived from rabbit, were infected with Sendai virus at 10 PFU/cell. 4 h later, ascites containing equivalent ELISA titers of IgA specific for the viral HN protein (clone 37 HN; A ), IgG specific for the viral HN protein (clone 20 HN; B ), or an irrelevant IgA (mineral oil plasmacytoma line 315; C ) were added to the basolateral surface as previously described ( 6 ). Productive viral infection is apparent in cells treated with specific IgG ( B ) or irrelevant IgA ( C ), with dense accretions of viral protein in patches on the apical portion of the cytoplasmic membrane ( arrows ), sometimes forming domed buds containing fibrillar chromatin-like material ( arrowheads ). Note that in A and C , small (5 nm) gold particles label the viral proteins, whereas in B , viral protein is detected by large (15 nm) gold particles. Essentially no intracellular Ig is identified in these latter specimens. By contrast, infected cells treated with specific IgA ( A ) show little viral protein at the cell surface and no accretions of viral protein or bud formations. Bar = 0.5 μm.
Figure Legend Snippet: Treatment with specific IgA, but not specific IgG or irrelevant IgA, reduces the appearance of viral protein on the cell surface. Polarized monolayers of MDCK cells in culture well inserts, stably transfected with the pIgR derived from rabbit, were infected with Sendai virus at 10 PFU/cell. 4 h later, ascites containing equivalent ELISA titers of IgA specific for the viral HN protein (clone 37 HN; A ), IgG specific for the viral HN protein (clone 20 HN; B ), or an irrelevant IgA (mineral oil plasmacytoma line 315; C ) were added to the basolateral surface as previously described ( 6 ). Productive viral infection is apparent in cells treated with specific IgG ( B ) or irrelevant IgA ( C ), with dense accretions of viral protein in patches on the apical portion of the cytoplasmic membrane ( arrows ), sometimes forming domed buds containing fibrillar chromatin-like material ( arrowheads ). Note that in A and C , small (5 nm) gold particles label the viral proteins, whereas in B , viral protein is detected by large (15 nm) gold particles. Essentially no intracellular Ig is identified in these latter specimens. By contrast, infected cells treated with specific IgA ( A ) show little viral protein at the cell surface and no accretions of viral protein or bud formations. Bar = 0.5 μm.

Techniques Used: Stable Transfection, Transfection, Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay

Specific IgA promotes the formation of membrane-delimited inclusions of multiple palisaded layers. Polarized MDCK cells were infected with 30 PFU/cell and 4 h later were incubated with 15 nm gold-labeled IgA anti-HN ( A ), IgG (data not shown), or irrelevant IgA ( B ), applied basolaterally. Other (control) polarized MDCK cells were also infected with 30 PFU/cell but 4 h later were treated basolaterally with unlabeled IgA anti-HN ( C ) or apically with 15 nm gold-labeled IgA anti-HN ( D ). Cells treated basolaterally with gold-labeled IgA anti-HN contain aggregates of numerous gold particles within numerous vesicular structures ( A ) that contain multi-lamellar structures ( A , inset ). In contrast, although a few gold particles are seen within vesicles in cells treated with IgG anti-HN (data not shown) or irrelevant IgA ( B ), the massive aggregates of gold associated with multilamellar structures, seen in A , are not visible. Infected cells treated with unlabeled IgA anti-HN ( C ) demonstrate vesicles similar in appearance to those in cells receiving gold-labeled specific IgA ( A ) but without gold particles, indicating that the formation of these structures is not due to the presence of gold. Although infected cells apically treated with gold-labeled IgA anti-HN exhibit a few gold particles in vesicles ( D ), large aggregates of gold and inclusions containing lamellae are not visualized, indicating that the initial reaction between IgA antibody and viral protein occurs within the cell during transcytosis and not near the cell surface after release into the apical supernatant upon subsequent re-uptake. Bars: A , 1 μm; inset to A , 0.25 μm; B–D , 0.5 μm.
Figure Legend Snippet: Specific IgA promotes the formation of membrane-delimited inclusions of multiple palisaded layers. Polarized MDCK cells were infected with 30 PFU/cell and 4 h later were incubated with 15 nm gold-labeled IgA anti-HN ( A ), IgG (data not shown), or irrelevant IgA ( B ), applied basolaterally. Other (control) polarized MDCK cells were also infected with 30 PFU/cell but 4 h later were treated basolaterally with unlabeled IgA anti-HN ( C ) or apically with 15 nm gold-labeled IgA anti-HN ( D ). Cells treated basolaterally with gold-labeled IgA anti-HN contain aggregates of numerous gold particles within numerous vesicular structures ( A ) that contain multi-lamellar structures ( A , inset ). In contrast, although a few gold particles are seen within vesicles in cells treated with IgG anti-HN (data not shown) or irrelevant IgA ( B ), the massive aggregates of gold associated with multilamellar structures, seen in A , are not visible. Infected cells treated with unlabeled IgA anti-HN ( C ) demonstrate vesicles similar in appearance to those in cells receiving gold-labeled specific IgA ( A ) but without gold particles, indicating that the formation of these structures is not due to the presence of gold. Although infected cells apically treated with gold-labeled IgA anti-HN exhibit a few gold particles in vesicles ( D ), large aggregates of gold and inclusions containing lamellae are not visualized, indicating that the initial reaction between IgA antibody and viral protein occurs within the cell during transcytosis and not near the cell surface after release into the apical supernatant upon subsequent re-uptake. Bars: A , 1 μm; inset to A , 0.25 μm; B–D , 0.5 μm.

Techniques Used: Infection, Incubation, Labeling

18) Product Images from "Immunocytochemical Colocalization of Specific Immunoglobulin A with Sendai Virus Protein in Infected Polarized Epithelium "

Article Title: Immunocytochemical Colocalization of Specific Immunoglobulin A with Sendai Virus Protein in Infected Polarized Epithelium

Journal: The Journal of Experimental Medicine

doi:

Colocalization of specific IgA and viral protein within the cytoplasm of Sendai virus–infected cells. In polarized MDCK cells infected with Sendai virus and treated by basolateral application of IgA anti-HN 4 h later ( A , a replicate culture of Fig. 1 A ), there is colocalization of numerous large (15 nm) gold particles labeling IgA ( large arrows ) and numerous small (5 nm) gold particles labeling viral protein ( small arrows ) to form multilamellar membrane structures, located deep within the cytosol. In infected cells treated with specific IgG ( B ) or irrelevant IgA ( C ), budding virions arising from the cell surface (indicated by asterisks) are identified by anti-viral HN protein staining (15-nm gold particles in B , large arrow ; 5-nm gold particles in C , small arrow ). The intracellular inclusions which develop in infected cells treated with specific IgA are only rarely identified in cells treated with specific IgG, and Ig does not frequently colocalize with viral protein. Neither infected cells treated with irrelevant IgA nor uninfected cells ever contain these structures. Bar = 0.25 μm.
Figure Legend Snippet: Colocalization of specific IgA and viral protein within the cytoplasm of Sendai virus–infected cells. In polarized MDCK cells infected with Sendai virus and treated by basolateral application of IgA anti-HN 4 h later ( A , a replicate culture of Fig. 1 A ), there is colocalization of numerous large (15 nm) gold particles labeling IgA ( large arrows ) and numerous small (5 nm) gold particles labeling viral protein ( small arrows ) to form multilamellar membrane structures, located deep within the cytosol. In infected cells treated with specific IgG ( B ) or irrelevant IgA ( C ), budding virions arising from the cell surface (indicated by asterisks) are identified by anti-viral HN protein staining (15-nm gold particles in B , large arrow ; 5-nm gold particles in C , small arrow ). The intracellular inclusions which develop in infected cells treated with specific IgA are only rarely identified in cells treated with specific IgG, and Ig does not frequently colocalize with viral protein. Neither infected cells treated with irrelevant IgA nor uninfected cells ever contain these structures. Bar = 0.25 μm.

Techniques Used: Infection, Labeling, Staining

Treatment with specific IgA, but not specific IgG or irrelevant IgA, reduces the appearance of viral protein on the cell surface. Polarized monolayers of MDCK cells in culture well inserts, stably transfected with the pIgR derived from rabbit, were infected with Sendai virus at 10 PFU/cell. 4 h later, ascites containing equivalent ELISA titers of IgA specific for the viral HN protein (clone 37 HN; A ), IgG specific for the viral HN protein (clone 20 HN; B ), or an irrelevant IgA (mineral oil plasmacytoma line 315; C ) were added to the basolateral surface as previously described ( 6 ). Productive viral infection is apparent in cells treated with specific IgG ( B ) or irrelevant IgA ( C ), with dense accretions of viral protein in patches on the apical portion of the cytoplasmic membrane ( arrows ), sometimes forming domed buds containing fibrillar chromatin-like material ( arrowheads ). Note that in A and C , small (5 nm) gold particles label the viral proteins, whereas in B , viral protein is detected by large (15 nm) gold particles. Essentially no intracellular Ig is identified in these latter specimens. By contrast, infected cells treated with specific IgA ( A ) show little viral protein at the cell surface and no accretions of viral protein or bud formations. Bar = 0.5 μm.
Figure Legend Snippet: Treatment with specific IgA, but not specific IgG or irrelevant IgA, reduces the appearance of viral protein on the cell surface. Polarized monolayers of MDCK cells in culture well inserts, stably transfected with the pIgR derived from rabbit, were infected with Sendai virus at 10 PFU/cell. 4 h later, ascites containing equivalent ELISA titers of IgA specific for the viral HN protein (clone 37 HN; A ), IgG specific for the viral HN protein (clone 20 HN; B ), or an irrelevant IgA (mineral oil plasmacytoma line 315; C ) were added to the basolateral surface as previously described ( 6 ). Productive viral infection is apparent in cells treated with specific IgG ( B ) or irrelevant IgA ( C ), with dense accretions of viral protein in patches on the apical portion of the cytoplasmic membrane ( arrows ), sometimes forming domed buds containing fibrillar chromatin-like material ( arrowheads ). Note that in A and C , small (5 nm) gold particles label the viral proteins, whereas in B , viral protein is detected by large (15 nm) gold particles. Essentially no intracellular Ig is identified in these latter specimens. By contrast, infected cells treated with specific IgA ( A ) show little viral protein at the cell surface and no accretions of viral protein or bud formations. Bar = 0.5 μm.

Techniques Used: Stable Transfection, Transfection, Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay

Specific IgA promotes the formation of membrane-delimited inclusions of multiple palisaded layers. Polarized MDCK cells were infected with 30 PFU/cell and 4 h later were incubated with 15 nm gold-labeled IgA anti-HN ( A ), IgG (data not shown), or irrelevant IgA ( B ), applied basolaterally. Other (control) polarized MDCK cells were also infected with 30 PFU/cell but 4 h later were treated basolaterally with unlabeled IgA anti-HN ( C ) or apically with 15 nm gold-labeled IgA anti-HN ( D ). Cells treated basolaterally with gold-labeled IgA anti-HN contain aggregates of numerous gold particles within numerous vesicular structures ( A ) that contain multi-lamellar structures ( A , inset ). In contrast, although a few gold particles are seen within vesicles in cells treated with IgG anti-HN (data not shown) or irrelevant IgA ( B ), the massive aggregates of gold associated with multilamellar structures, seen in A , are not visible. Infected cells treated with unlabeled IgA anti-HN ( C ) demonstrate vesicles similar in appearance to those in cells receiving gold-labeled specific IgA ( A ) but without gold particles, indicating that the formation of these structures is not due to the presence of gold. Although infected cells apically treated with gold-labeled IgA anti-HN exhibit a few gold particles in vesicles ( D ), large aggregates of gold and inclusions containing lamellae are not visualized, indicating that the initial reaction between IgA antibody and viral protein occurs within the cell during transcytosis and not near the cell surface after release into the apical supernatant upon subsequent re-uptake. Bars: A , 1 μm; inset to A , 0.25 μm; B–D , 0.5 μm.
Figure Legend Snippet: Specific IgA promotes the formation of membrane-delimited inclusions of multiple palisaded layers. Polarized MDCK cells were infected with 30 PFU/cell and 4 h later were incubated with 15 nm gold-labeled IgA anti-HN ( A ), IgG (data not shown), or irrelevant IgA ( B ), applied basolaterally. Other (control) polarized MDCK cells were also infected with 30 PFU/cell but 4 h later were treated basolaterally with unlabeled IgA anti-HN ( C ) or apically with 15 nm gold-labeled IgA anti-HN ( D ). Cells treated basolaterally with gold-labeled IgA anti-HN contain aggregates of numerous gold particles within numerous vesicular structures ( A ) that contain multi-lamellar structures ( A , inset ). In contrast, although a few gold particles are seen within vesicles in cells treated with IgG anti-HN (data not shown) or irrelevant IgA ( B ), the massive aggregates of gold associated with multilamellar structures, seen in A , are not visible. Infected cells treated with unlabeled IgA anti-HN ( C ) demonstrate vesicles similar in appearance to those in cells receiving gold-labeled specific IgA ( A ) but without gold particles, indicating that the formation of these structures is not due to the presence of gold. Although infected cells apically treated with gold-labeled IgA anti-HN exhibit a few gold particles in vesicles ( D ), large aggregates of gold and inclusions containing lamellae are not visualized, indicating that the initial reaction between IgA antibody and viral protein occurs within the cell during transcytosis and not near the cell surface after release into the apical supernatant upon subsequent re-uptake. Bars: A , 1 μm; inset to A , 0.25 μm; B–D , 0.5 μm.

Techniques Used: Infection, Incubation, Labeling

19) Product Images from "Bronchus-associated lymphoid tissue–resident Foxp3+ T lymphocytes prevent antibody-mediated lung rejection"

Article Title: Bronchus-associated lymphoid tissue–resident Foxp3+ T lymphocytes prevent antibody-mediated lung rejection

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI122083

AMR after depletion of graft-resident Foxp3 + T cells is dependent on graft infiltration by T cells. Plots and histograms depicting markers characteristic of Tfh cells on CD4 + T cells derived from ( A and C ) primary (CD45.2) or ( B and D ) secondary (CD45.1) recipients in BALB/c lungs, initially transplanted into immunosuppressed ( A and B ) B6 (CD45.2 + ) or ( C and D ) B6 Foxp3-DTR (CD45.2 + ) mice and, at least 30 days later, retransplanted into DT-treated B6 (CD45.1 + ) hosts. Plots are gated on live CD45.2 + CD45.1 – CD90.2 + CD4 + CD8 – Foxp3 – and CD45.2 + CD45.1 + CD90.2 + CD4 + CD8 – Foxp3 – cells. Histograms are gated on CD4 + T cells that are PD-1 hi CXCR5 + (bcl-6, red; isotype control, blue) ( n = 4 each). Quantification of ( E ) CD45.2 + and ( F ) CD45.1 + CD4 + T cells that are PD-1 hi CXCR5 + in (circles) control and (inverted triangles) Foxp3 + T cell–depleted lungs 7 days after retransplantation. ( G ) Gross and ( H ) histological appearance (H E) and staining for ( I ) MT, ( J ) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 nude hosts. Scale bars: 100 μm. ( K ) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or nude (red) B6 hosts at least 30 days after initial engraftment into immunosuppressed B6 Foxp3-DTR mice. Data are expressed as mean ± SEM ( n = 4 mice per group). Mann-Whitney U test was used to compare the means.
Figure Legend Snippet: AMR after depletion of graft-resident Foxp3 + T cells is dependent on graft infiltration by T cells. Plots and histograms depicting markers characteristic of Tfh cells on CD4 + T cells derived from ( A and C ) primary (CD45.2) or ( B and D ) secondary (CD45.1) recipients in BALB/c lungs, initially transplanted into immunosuppressed ( A and B ) B6 (CD45.2 + ) or ( C and D ) B6 Foxp3-DTR (CD45.2 + ) mice and, at least 30 days later, retransplanted into DT-treated B6 (CD45.1 + ) hosts. Plots are gated on live CD45.2 + CD45.1 – CD90.2 + CD4 + CD8 – Foxp3 – and CD45.2 + CD45.1 + CD90.2 + CD4 + CD8 – Foxp3 – cells. Histograms are gated on CD4 + T cells that are PD-1 hi CXCR5 + (bcl-6, red; isotype control, blue) ( n = 4 each). Quantification of ( E ) CD45.2 + and ( F ) CD45.1 + CD4 + T cells that are PD-1 hi CXCR5 + in (circles) control and (inverted triangles) Foxp3 + T cell–depleted lungs 7 days after retransplantation. ( G ) Gross and ( H ) histological appearance (H E) and staining for ( I ) MT, ( J ) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 nude hosts. Scale bars: 100 μm. ( K ) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or nude (red) B6 hosts at least 30 days after initial engraftment into immunosuppressed B6 Foxp3-DTR mice. Data are expressed as mean ± SEM ( n = 4 mice per group). Mann-Whitney U test was used to compare the means.

Techniques Used: Derivative Assay, Mouse Assay, Staining, Activated Clotting Time Assay, MANN-WHITNEY

AMR of Foxp3 + T cell–depleted lung grafts can be prevented by blocking ICOS/ICOS ligand and CD40/CD40 ligand pathways. ( A ) Expression levels of ICOS, ICOS ligand (ICOSL), CD40, CD40 ligand (CD40L), and IL-21 in BALB/c lung grafts, which were initially transplanted into immunosuppressed B6 WT (CD45.2) (circles) or Foxp3-DTR B6 (CD45.2) (inverted triangles) mice and at least 30 days later retransplanted into DT-treated (days 0 and 1) B6 CD45.1 hosts. Grafts were examined 7 days after retransplantation ( n = 5 each). ** P
Figure Legend Snippet: AMR of Foxp3 + T cell–depleted lung grafts can be prevented by blocking ICOS/ICOS ligand and CD40/CD40 ligand pathways. ( A ) Expression levels of ICOS, ICOS ligand (ICOSL), CD40, CD40 ligand (CD40L), and IL-21 in BALB/c lung grafts, which were initially transplanted into immunosuppressed B6 WT (CD45.2) (circles) or Foxp3-DTR B6 (CD45.2) (inverted triangles) mice and at least 30 days later retransplanted into DT-treated (days 0 and 1) B6 CD45.1 hosts. Grafts were examined 7 days after retransplantation ( n = 5 each). ** P

Techniques Used: Blocking Assay, Expressing, Mouse Assay

Depletion of graft-resident Foxp3 + T cells triggers AMR. ( A ) Schematic depicting experimental model. CSB, costimulation blockade. Plots and quantification of CD4 + Foxp3 + cells from ( B ) primary (CD45.2) or ( C ) secondary recipient (CD45.1) in BALB/c lungs, transplanted into immunosuppressed WT B6 (CD45.2) (circles) or Foxp3-DTR B6 (CD45.2) (inverted triangles) mice and retransplanted into DT-treated B6 (CD45.1) hosts at least 30 days later. Plots are gated on live CD45.2 + CD45.1 – CD90.2 + and live CD45.2 – CD45.1 + CD90.2 + cells. ( D ) Plots and quantification of distribution of CD45.1 vs. CD45.2 on live CD90.2 + CD4 + CD8 – Foxp3 + cells without and with depletion of graft-resident (CD45.2) Foxp3 + cells. Gross and histological appearance (H E) of BALB/c lungs, transplanted into immunosuppressed WT ( E and G ) or Foxp3-DTR ( F and H ) B6 CD45.2 + mice and, at least 30 days later, retransplanted into DT-treated B6 CD45.1 + hosts ( n = 4 mice per group). ( I ) Airway epithelium (arrow) in human lung diagnosed with AMR (H E) ( n = 11). Staining of ( J and K ) MT (blue), ( L and M ) CCSP (red), AcT (green), and ( N and O ) C4d (brown) in BALB/c lungs, transplanted into immunosuppressed ( J , L , and N ) WT or ( K , M , and O ) Foxp3-DTR B6 CD45.2 + mice and, at least 30 days later, retransplanted into DT-treated B6 CD45.1 + hosts. Scale bars: 100 μm. Arrows in G , J , and L point to BALT. ( P ) Donor-specific IgM antibody titers 7 days after retransplantation of BALB/c lungs into DT-treated B6 CD45.1 + hosts at least 30 days after initial engraftment into immunosuppressed B6 WT (red) or B6 Foxp3-DTR (blue) mice. ( Q ) Reactivity of serum IgM antibodies, following depletion of graft-resident Foxp3 cells, against donor (BALB/c), recipient (B6), and third-party (CBA) antigen ( n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.
Figure Legend Snippet: Depletion of graft-resident Foxp3 + T cells triggers AMR. ( A ) Schematic depicting experimental model. CSB, costimulation blockade. Plots and quantification of CD4 + Foxp3 + cells from ( B ) primary (CD45.2) or ( C ) secondary recipient (CD45.1) in BALB/c lungs, transplanted into immunosuppressed WT B6 (CD45.2) (circles) or Foxp3-DTR B6 (CD45.2) (inverted triangles) mice and retransplanted into DT-treated B6 (CD45.1) hosts at least 30 days later. Plots are gated on live CD45.2 + CD45.1 – CD90.2 + and live CD45.2 – CD45.1 + CD90.2 + cells. ( D ) Plots and quantification of distribution of CD45.1 vs. CD45.2 on live CD90.2 + CD4 + CD8 – Foxp3 + cells without and with depletion of graft-resident (CD45.2) Foxp3 + cells. Gross and histological appearance (H E) of BALB/c lungs, transplanted into immunosuppressed WT ( E and G ) or Foxp3-DTR ( F and H ) B6 CD45.2 + mice and, at least 30 days later, retransplanted into DT-treated B6 CD45.1 + hosts ( n = 4 mice per group). ( I ) Airway epithelium (arrow) in human lung diagnosed with AMR (H E) ( n = 11). Staining of ( J and K ) MT (blue), ( L and M ) CCSP (red), AcT (green), and ( N and O ) C4d (brown) in BALB/c lungs, transplanted into immunosuppressed ( J , L , and N ) WT or ( K , M , and O ) Foxp3-DTR B6 CD45.2 + mice and, at least 30 days later, retransplanted into DT-treated B6 CD45.1 + hosts. Scale bars: 100 μm. Arrows in G , J , and L point to BALT. ( P ) Donor-specific IgM antibody titers 7 days after retransplantation of BALB/c lungs into DT-treated B6 CD45.1 + hosts at least 30 days after initial engraftment into immunosuppressed B6 WT (red) or B6 Foxp3-DTR (blue) mice. ( Q ) Reactivity of serum IgM antibodies, following depletion of graft-resident Foxp3 cells, against donor (BALB/c), recipient (B6), and third-party (CBA) antigen ( n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.

Techniques Used: Mouse Assay, Staining, Activated Clotting Time Assay, Crocin Bleaching Assay, MANN-WHITNEY

Graft-resident Foxp3 + T cells maintain lung transplant tolerance. ( A and E ) Gross and ( B and F ) histological appearance (H E), ( C and G ) MT staining (blue), and ( D and H ) immunofluorescent staining of CCSP (red) and AcT (green) in BALB/c lungs, initially transplanted into immunosuppressed ( A – D ) B6 (CD45.2 + ) or ( E – H ) B6 Foxp3-DTR (CD45.2 + ) mice and retransplanted into DT-treated secondary B6 (CD45.1 + ) hosts at least 30 days after primary transplantation. Secondary hosts were examined 30 days after retransplantation. Scale bars: 100 μm. Arrows in B and C point to BALT. Serum titers of donor-specific ( I ) IgM and ( J ) IgG antibodies 30 days after retransplantation of BALB/c lungs, initially transplanted into immunosuppressed B6 WT (red) or B6 Foxp3-DTR (blue) mice and, at least 30 days later, retransplanted into B6 45.1 + hosts. ( K ) Reactivity of serum IgG antibodies, following depletion of graft-resident Foxp3 cells, against donor (BALB/c), recipient (B6), and third-party (CBA) antigen. Data are expressed as mean ± SEM ( n = 4 mice per group). Mann-Whitney U test was used to compare the means.
Figure Legend Snippet: Graft-resident Foxp3 + T cells maintain lung transplant tolerance. ( A and E ) Gross and ( B and F ) histological appearance (H E), ( C and G ) MT staining (blue), and ( D and H ) immunofluorescent staining of CCSP (red) and AcT (green) in BALB/c lungs, initially transplanted into immunosuppressed ( A – D ) B6 (CD45.2 + ) or ( E – H ) B6 Foxp3-DTR (CD45.2 + ) mice and retransplanted into DT-treated secondary B6 (CD45.1 + ) hosts at least 30 days after primary transplantation. Secondary hosts were examined 30 days after retransplantation. Scale bars: 100 μm. Arrows in B and C point to BALT. Serum titers of donor-specific ( I ) IgM and ( J ) IgG antibodies 30 days after retransplantation of BALB/c lungs, initially transplanted into immunosuppressed B6 WT (red) or B6 Foxp3-DTR (blue) mice and, at least 30 days later, retransplanted into B6 45.1 + hosts. ( K ) Reactivity of serum IgG antibodies, following depletion of graft-resident Foxp3 cells, against donor (BALB/c), recipient (B6), and third-party (CBA) antigen. Data are expressed as mean ± SEM ( n = 4 mice per group). Mann-Whitney U test was used to compare the means.

Techniques Used: Staining, Activated Clotting Time Assay, Mouse Assay, Transplantation Assay, Crocin Bleaching Assay, MANN-WHITNEY

Graft-infiltrating B cells trigger AMR after depletion of graft-resident Foxp3 + T cells. Activated B cells from ( A and B ) primary recipient (CD45.2) or ( C and D ) secondary recipient (CD45.1) in BALB/c lungs, transplanted into immunosuppressed ( A and C ) B6 (CD45.2 + ) (no Foxp3 depletion) or ( B and D ) B6 Foxp3-DTR (CD45.2 + ) mice (Foxp3 depletion) and, at least 30 days later, retransplanted into DT-treated B6 (CD45.1 + ) hosts. Plots are gated on live CD45.2 + CD45.1 – B220 + (donor) and live CD45.2 – CD45.1 + B220 + cells (recipient). Quantification of activated ( E ) CD45.2 and ( F ) CD45.1 B cells in (circles) control and (inverted triangles) Foxp3 + T cell–depleted lungs 7 days after retransplantation. ( G ) Gross, ( H ) histological appearance (H E), ( I ) MT staining (blue), and ( J ) CCSP (red) and AcT (green) staining in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 muMt – hosts. ( K ) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or muMt – (red) B6 hosts at least 30 days after engraftment into immunosuppressed B6 Foxp3-DTR mice. ( L ) Gross and ( M ) histological appearance (H E) of BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 AID/μS knockout hosts. Scale bars: 100 μm. ( N ) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or AID/μS knockout (red) B6 hosts at least 30 days after engraftment into immunosuppressed B6 Foxp3-DTR mice. Data are expressed as mean ± SEM ( n = 4 mice per group). Mann-Whitney U test was used to compare the means.
Figure Legend Snippet: Graft-infiltrating B cells trigger AMR after depletion of graft-resident Foxp3 + T cells. Activated B cells from ( A and B ) primary recipient (CD45.2) or ( C and D ) secondary recipient (CD45.1) in BALB/c lungs, transplanted into immunosuppressed ( A and C ) B6 (CD45.2 + ) (no Foxp3 depletion) or ( B and D ) B6 Foxp3-DTR (CD45.2 + ) mice (Foxp3 depletion) and, at least 30 days later, retransplanted into DT-treated B6 (CD45.1 + ) hosts. Plots are gated on live CD45.2 + CD45.1 – B220 + (donor) and live CD45.2 – CD45.1 + B220 + cells (recipient). Quantification of activated ( E ) CD45.2 and ( F ) CD45.1 B cells in (circles) control and (inverted triangles) Foxp3 + T cell–depleted lungs 7 days after retransplantation. ( G ) Gross, ( H ) histological appearance (H E), ( I ) MT staining (blue), and ( J ) CCSP (red) and AcT (green) staining in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 muMt – hosts. ( K ) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or muMt – (red) B6 hosts at least 30 days after engraftment into immunosuppressed B6 Foxp3-DTR mice. ( L ) Gross and ( M ) histological appearance (H E) of BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 AID/μS knockout hosts. Scale bars: 100 μm. ( N ) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or AID/μS knockout (red) B6 hosts at least 30 days after engraftment into immunosuppressed B6 Foxp3-DTR mice. Data are expressed as mean ± SEM ( n = 4 mice per group). Mann-Whitney U test was used to compare the means.

Techniques Used: Mouse Assay, Staining, Activated Clotting Time Assay, Knock-Out, MANN-WHITNEY

Foxp3 + T lymphocyte depletion–triggered AMR is dependent on CXCL13-mediated chemokinesis. ( A ) Foxp3 + (green), B cells (blue), and CD4 + T cells (red) in BALB/c lungs at least 30 days after transplantation into immunosuppressed B6 Foxp3-IRES GFP recipient ( n = 3). Scale bar: 10 μm. CXCR5 + B cells from secondary host (recipient) in BALB/c lungs, initially transplanted into immunosuppressed ( B ) WT or ( C ) Foxp3-DTR B6 (CD45.2 + ) recipient and, at least 30 days later, retransplanted into DT-treated B6 CD45.1 + hosts. Plots are gated on live CD45.2 – CD45.1 + cells. ( D ) CD45.1 + CXCR5 + B cells in (circles) control and (inverted triangles) Foxp3 + T cell–depleted lungs 7 days after retransplantation ( n = 4 each). CXCL13 (brown) in ( E ) control and ( F ) Foxp3 + T cell–depleted grafts 7 days after retransplantation. Scale bars: 100 μm. CD4 + T cells (green) and B cells (blue) in BALB/c lungs, initially transplanted into immunosuppressed B6 Foxp3-DTR recipients and, at least 30 days later, retransplanted into B6 hosts, treated with ( G ) DT/control-Ig (arrows: CD4 + T–B cell interactions) or ( H ) DT/anti-CXCL13 ( n = 2 each) (red, quantum dots). Scale bars: 20 μm. ( I ) Contact duration between CD4 + T and B cells, ( J ) CD4 + T, and ( K ) B cell mean square displacements and ( L ) CD4 + T and ( M ) B cell velocities within retransplanted Foxp3 + T cell–depleted BALB/c lungs with and without CXCL13 inhibition. ( N ) Gross, ( O ) histological appearance (H E), staining for ( P ) MT (blue), ( Q ) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT- and anti-CXCL13–treated B6 hosts. Scale bars: 100 μm. ( R ) Donor-specific IgM titers after retransplantation of BALB/c lungs into DT-treated control (blue) or DT/anti-CXCL13 antibody–treated (red) B6 recipients after initial engraftment into immunosuppressed B6 Foxp3-DTR mice ( n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.
Figure Legend Snippet: Foxp3 + T lymphocyte depletion–triggered AMR is dependent on CXCL13-mediated chemokinesis. ( A ) Foxp3 + (green), B cells (blue), and CD4 + T cells (red) in BALB/c lungs at least 30 days after transplantation into immunosuppressed B6 Foxp3-IRES GFP recipient ( n = 3). Scale bar: 10 μm. CXCR5 + B cells from secondary host (recipient) in BALB/c lungs, initially transplanted into immunosuppressed ( B ) WT or ( C ) Foxp3-DTR B6 (CD45.2 + ) recipient and, at least 30 days later, retransplanted into DT-treated B6 CD45.1 + hosts. Plots are gated on live CD45.2 – CD45.1 + cells. ( D ) CD45.1 + CXCR5 + B cells in (circles) control and (inverted triangles) Foxp3 + T cell–depleted lungs 7 days after retransplantation ( n = 4 each). CXCL13 (brown) in ( E ) control and ( F ) Foxp3 + T cell–depleted grafts 7 days after retransplantation. Scale bars: 100 μm. CD4 + T cells (green) and B cells (blue) in BALB/c lungs, initially transplanted into immunosuppressed B6 Foxp3-DTR recipients and, at least 30 days later, retransplanted into B6 hosts, treated with ( G ) DT/control-Ig (arrows: CD4 + T–B cell interactions) or ( H ) DT/anti-CXCL13 ( n = 2 each) (red, quantum dots). Scale bars: 20 μm. ( I ) Contact duration between CD4 + T and B cells, ( J ) CD4 + T, and ( K ) B cell mean square displacements and ( L ) CD4 + T and ( M ) B cell velocities within retransplanted Foxp3 + T cell–depleted BALB/c lungs with and without CXCL13 inhibition. ( N ) Gross, ( O ) histological appearance (H E), staining for ( P ) MT (blue), ( Q ) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT- and anti-CXCL13–treated B6 hosts. Scale bars: 100 μm. ( R ) Donor-specific IgM titers after retransplantation of BALB/c lungs into DT-treated control (blue) or DT/anti-CXCL13 antibody–treated (red) B6 recipients after initial engraftment into immunosuppressed B6 Foxp3-DTR mice ( n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.

Techniques Used: Transplantation Assay, Inhibition, Staining, Activated Clotting Time Assay, Mouse Assay, MANN-WHITNEY

Long-term acceptance after lung transplantation is associated with induction of Foxp3 + cell–rich BALT. ( A ) Gross and ( B ) histological appearance (H E) of BALB/c lung graft (Tx) at least 30 days after transplantation into an immunosuppressed B6 host. Arrow depicts induced BALT ( n = 8). Scale bar: 100 μm. ( C ) PNAd staining (brown), ( D ) MT staining (blue), and ( E ) immunofluorescent staining of CCSP (red) and AcT (green) in BALB/c lung graft at least 30 days after transplantation into an immunosuppressed B6 host. Scale bars: 100 μm. ( F ) Intravital 2-photon (2P) imaging depicting aggregates of Foxp3 + cells in BALB/c lung graft at least 30 days after transplantation into an immunosuppressed B6 Foxp3-IRES GFP recipient (Foxp3 + cells, green; quantum dot–labeled vessels, red) ( n = 3). Scale bar: 30 μm.
Figure Legend Snippet: Long-term acceptance after lung transplantation is associated with induction of Foxp3 + cell–rich BALT. ( A ) Gross and ( B ) histological appearance (H E) of BALB/c lung graft (Tx) at least 30 days after transplantation into an immunosuppressed B6 host. Arrow depicts induced BALT ( n = 8). Scale bar: 100 μm. ( C ) PNAd staining (brown), ( D ) MT staining (blue), and ( E ) immunofluorescent staining of CCSP (red) and AcT (green) in BALB/c lung graft at least 30 days after transplantation into an immunosuppressed B6 host. Scale bars: 100 μm. ( F ) Intravital 2-photon (2P) imaging depicting aggregates of Foxp3 + cells in BALB/c lung graft at least 30 days after transplantation into an immunosuppressed B6 Foxp3-IRES GFP recipient (Foxp3 + cells, green; quantum dot–labeled vessels, red) ( n = 3). Scale bar: 30 μm.

Techniques Used: Transplantation Assay, Staining, Activated Clotting Time Assay, Imaging, Labeling

20) Product Images from "The Inner Foreskin of Healthy Males at Risk of HIV Infection Harbors Epithelial CD4+ CCR5+ Cells and Has Features of an Inflamed Epidermal Barrier"

Article Title: The Inner Foreskin of Healthy Males at Risk of HIV Infection Harbors Epithelial CD4+ CCR5+ Cells and Has Features of an Inflamed Epidermal Barrier

Journal: PLoS ONE

doi: 10.1371/journal.pone.0108954

Tight junction proteins differentially accumulate in the inner and outer foreskin sections from sexually active men. Samples from 11 participants were selected for immunofluorescence studies. A, D, G) Representative images of foreskin epidermis at 40× magnification stained with A) claudin 1, D) claudin 4, and G) occludin (pseudocolored green) or their isotype controls. All the fields in ∼26.5 mm 2 of tissue per participant were used for analysis. B, E, H) Percent of epidermal area covered by B) claudin 1, E) claudin 4 or H) occludin stain. C, I) Mean intensity of C) claudin 1 or I) occludin within the epidermis. F) The angular second moment of claudin 4 staining within foreskin epidermis. All p-values are from FDR-adjusted Wilcoxon tests.
Figure Legend Snippet: Tight junction proteins differentially accumulate in the inner and outer foreskin sections from sexually active men. Samples from 11 participants were selected for immunofluorescence studies. A, D, G) Representative images of foreskin epidermis at 40× magnification stained with A) claudin 1, D) claudin 4, and G) occludin (pseudocolored green) or their isotype controls. All the fields in ∼26.5 mm 2 of tissue per participant were used for analysis. B, E, H) Percent of epidermal area covered by B) claudin 1, E) claudin 4 or H) occludin stain. C, I) Mean intensity of C) claudin 1 or I) occludin within the epidermis. F) The angular second moment of claudin 4 staining within foreskin epidermis. All p-values are from FDR-adjusted Wilcoxon tests.

Techniques Used: Immunofluorescence, Staining

21) Product Images from "The Localization of the Brain-Specific Inorganic Phosphate Transporter Suggests a Specific Presynaptic Role in Glutamatergic Transmission"

Article Title: The Localization of the Brain-Specific Inorganic Phosphate Transporter Suggests a Specific Presynaptic Role in Glutamatergic Transmission

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.18-21-08648.1998

BNPI localizes to synaptic vesicles at asymmetric synapses by immunogold-silver electron microscopy. Immunogold-silver electron microscopy localizes BNPI in axon terminals that form asymmetric excitatory-type synapses ( open arrowheads ) with unlabeled dendritic shafts ( UD ) or spines ( US ) in the rat caudate putamen nucleus. A, Immunogold-silver deposits are seen in direct contact with many small synaptic vesicles ( SSV ) within the BNPI-labeled terminals. Several gold particles also directly contact the plasma membrane ( arrowheads ) but only in the vicinity of synaptic vesicles. B, Immunogold-silver labeling for BNPI is associated with SSVs in two axon terminals, one of which forms an asymmetric synapse ( open arrowhead ) with a spine from the unlabeled dendrite ( UD ). An adjacent unlabeled terminal ( UT ) also forms an asymmetric synaptic contact ( open arrowhead ) with the shaft of the same dendrite. Scale bars, 0.5 μm.
Figure Legend Snippet: BNPI localizes to synaptic vesicles at asymmetric synapses by immunogold-silver electron microscopy. Immunogold-silver electron microscopy localizes BNPI in axon terminals that form asymmetric excitatory-type synapses ( open arrowheads ) with unlabeled dendritic shafts ( UD ) or spines ( US ) in the rat caudate putamen nucleus. A, Immunogold-silver deposits are seen in direct contact with many small synaptic vesicles ( SSV ) within the BNPI-labeled terminals. Several gold particles also directly contact the plasma membrane ( arrowheads ) but only in the vicinity of synaptic vesicles. B, Immunogold-silver labeling for BNPI is associated with SSVs in two axon terminals, one of which forms an asymmetric synapse ( open arrowhead ) with a spine from the unlabeled dendrite ( UD ). An adjacent unlabeled terminal ( UT ) also forms an asymmetric synaptic contact ( open arrowhead ) with the shaft of the same dendrite. Scale bars, 0.5 μm.

Techniques Used: Electron Microscopy, Labeling

22) Product Images from "P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation"

Article Title: P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation

Journal: Purinergic Signalling

doi: 10.1007/s11302-011-9235-x

Modulation of astroglial proliferation by nucleotides in highly enriched astroglial cultures ( a ,  c ) and co-cultures ( b ,  d ). Cultures were incubated with nucleotides or solvent for 48 h and  methyl -[ 3 H]-thymidine (1 μCi/ml) was added in the last 24 h. Cell proliferation was estimated by  methyl -[ 3 H]-thymidine incorporation and expressed in percentage of control. Values are means ± SEM from five to seven experiments. * P
Figure Legend Snippet: Modulation of astroglial proliferation by nucleotides in highly enriched astroglial cultures ( a , c ) and co-cultures ( b , d ). Cultures were incubated with nucleotides or solvent for 48 h and methyl -[ 3 H]-thymidine (1 μCi/ml) was added in the last 24 h. Cell proliferation was estimated by methyl -[ 3 H]-thymidine incorporation and expressed in percentage of control. Values are means ± SEM from five to seven experiments. * P

Techniques Used: Incubation

23) Product Images from "Iron oxide magnetic nanoparticles highlight early involvement of the choroid plexus in central nervous system inflammation"

Article Title: Iron oxide magnetic nanoparticles highlight early involvement of the choroid plexus in central nervous system inflammation

Journal: ASN NEURO

doi: 10.1042/AN20120081

VSOP observed in perivascular-restricted spinal cord lesions with intact BBB ( A ) GFAP immunostaining (brown) shows astrocyte end foot processes surrounding a lesion with immune infiltrates (stained with Nuclear Fast Red) and including VSOP (blue). ( B ) Immunostaining for laminin (brown) shows vascular endothelium and glia limitans of a perivascular lesion, along with infiltrating cells and VSOP (blue). ( C ) Immunostaining for iba-1 (brown) shows the presence of microglia in CNS parenchyma, and activated microglia/macrophages associated with VSOP (blue) located in the perivascular lesion. Original magnification: ( A – C ) ×200.
Figure Legend Snippet: VSOP observed in perivascular-restricted spinal cord lesions with intact BBB ( A ) GFAP immunostaining (brown) shows astrocyte end foot processes surrounding a lesion with immune infiltrates (stained with Nuclear Fast Red) and including VSOP (blue). ( B ) Immunostaining for laminin (brown) shows vascular endothelium and glia limitans of a perivascular lesion, along with infiltrating cells and VSOP (blue). ( C ) Immunostaining for iba-1 (brown) shows the presence of microglia in CNS parenchyma, and activated microglia/macrophages associated with VSOP (blue) located in the perivascular lesion. Original magnification: ( A – C ) ×200.

Techniques Used: Immunostaining, Staining

24) Product Images from "Neutrophil-derived JAML Inhibits Repair of Intestinal Epithelial Injury During Acute Inflammation"

Article Title: Neutrophil-derived JAML Inhibits Repair of Intestinal Epithelial Injury During Acute Inflammation

Journal: Mucosal immunology

doi: 10.1038/mi.2014.12

Characterization of an anti-human JAML mAb that inhibits JAML-CAR binding (a) Anti-JAML mAbs were added to Immulon plates coated with sJAML (JAML.D1D2), membrane distal (sJAML.D1) or membrane proximal (sJAML.D2) domains. Antibody binding was detected using goat anti-mouse HRP. M3 represents JAML antiserum collected from the sJAML-His immunized mouse before fusion. Anti-Myc mAb (9E10) was used as control. Both anti-JAML mAbs bound to the membrane distal domain (D1) of JAML. (b) mAbs to JAML (10 μg/ml) were added to plates coated with sJAML followed by addition of CAR-GST (5 μg/ml). CAR binding was detected by goat anti GST-HRP. DW100 inhibited CAR binding to sJAML, but DW216 had no effect. *** p
Figure Legend Snippet: Characterization of an anti-human JAML mAb that inhibits JAML-CAR binding (a) Anti-JAML mAbs were added to Immulon plates coated with sJAML (JAML.D1D2), membrane distal (sJAML.D1) or membrane proximal (sJAML.D2) domains. Antibody binding was detected using goat anti-mouse HRP. M3 represents JAML antiserum collected from the sJAML-His immunized mouse before fusion. Anti-Myc mAb (9E10) was used as control. Both anti-JAML mAbs bound to the membrane distal domain (D1) of JAML. (b) mAbs to JAML (10 μg/ml) were added to plates coated with sJAML followed by addition of CAR-GST (5 μg/ml). CAR binding was detected by goat anti GST-HRP. DW100 inhibited CAR binding to sJAML, but DW216 had no effect. *** p

Techniques Used: Binding Assay

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Recombinant:

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro
Article Snippet: .. Material used were endothelial basal medium (EBM) (CC-3121; Clonetics, San Diego, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); 100× GPS (29.2 mg/ml l -glutamine, 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate); gentamicin sulfate and 100× PSF (10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin B) (Life Technologies, Inc., Grand Island, NY); collagenase A (Boehringer Mannheim, Indianapolis, IN); Immobilon-P membrane (Millipore, Bedford, MA); Hyperfilm ECL, fluorescein-streptavidin, and Texas Red-streptavidin (Amersham Life Sciences, Arlington Heights, IL); Lumiglo (KPL); human TGF-β1, recombinant human TGF-β2 and -β3, recombinant human platelet-derived growth factor (PDGF)-BB, and anti-PDFG-BB (R & D Systems, Minneapolis, MN); Vectastain Elite ABC kit, avidin/biotin blocking kit, fluorescein anti-mouse IgG, Texas red anti-rabbit IgG, peroxidase-conjugated anti-goat IgG, biotinylated horse anti-mouse IgG, avidin-peroxidase, peroxidase-conjugated anti-mouse IgG, and 3,3′5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA); 3-amino-9-ethyl carbazol and mouse anti-human α-SMA (clone 1A4 ) (Sigma Chemical Co., St. Louis, MO); goat anti-human CD31/PECAM-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human von Willebrand factor (vWF) and mouse anti-human CD31/PECAM-1 (DAKO, Carpinteria, CA); polycarbonate PVP-F membranes (Neuro Probe, Inc, Gaithersburg, MD). .. Recombinant human bFGF was kindly provided by Scios Nova Inc., Mountain View, CA; soluble recombinant TGF-β type II receptor, prepared as described, was kindly provided by Philip Gotwals, Biogen, Cambridge, MA; SM1 antibody was kindly provided by Masanori Aikawa, Brigham and Women’s Hospital, Boston; rabbit anti-bovine CD31/PECAM-1 was kindly provided by Steven Albelda, University of Pennsylvania.

Avidin-Biotin Assay:

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro
Article Snippet: .. Material used were endothelial basal medium (EBM) (CC-3121; Clonetics, San Diego, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); 100× GPS (29.2 mg/ml l -glutamine, 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate); gentamicin sulfate and 100× PSF (10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin B) (Life Technologies, Inc., Grand Island, NY); collagenase A (Boehringer Mannheim, Indianapolis, IN); Immobilon-P membrane (Millipore, Bedford, MA); Hyperfilm ECL, fluorescein-streptavidin, and Texas Red-streptavidin (Amersham Life Sciences, Arlington Heights, IL); Lumiglo (KPL); human TGF-β1, recombinant human TGF-β2 and -β3, recombinant human platelet-derived growth factor (PDGF)-BB, and anti-PDFG-BB (R & D Systems, Minneapolis, MN); Vectastain Elite ABC kit, avidin/biotin blocking kit, fluorescein anti-mouse IgG, Texas red anti-rabbit IgG, peroxidase-conjugated anti-goat IgG, biotinylated horse anti-mouse IgG, avidin-peroxidase, peroxidase-conjugated anti-mouse IgG, and 3,3′5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA); 3-amino-9-ethyl carbazol and mouse anti-human α-SMA (clone 1A4 ) (Sigma Chemical Co., St. Louis, MO); goat anti-human CD31/PECAM-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human von Willebrand factor (vWF) and mouse anti-human CD31/PECAM-1 (DAKO, Carpinteria, CA); polycarbonate PVP-F membranes (Neuro Probe, Inc, Gaithersburg, MD). .. Recombinant human bFGF was kindly provided by Scios Nova Inc., Mountain View, CA; soluble recombinant TGF-β type II receptor, prepared as described, was kindly provided by Philip Gotwals, Biogen, Cambridge, MA; SM1 antibody was kindly provided by Masanori Aikawa, Brigham and Women’s Hospital, Boston; rabbit anti-bovine CD31/PECAM-1 was kindly provided by Steven Albelda, University of Pennsylvania.

Blocking Assay:

Article Title: Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-?-Mediated and Non-Transforming Growth Factor-?-Mediated Transdifferentiation in Vitro
Article Snippet: .. Material used were endothelial basal medium (EBM) (CC-3121; Clonetics, San Diego, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); 100× GPS (29.2 mg/ml l -glutamine, 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate); gentamicin sulfate and 100× PSF (10,000 U/ml penicillin G, 10,000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin B) (Life Technologies, Inc., Grand Island, NY); collagenase A (Boehringer Mannheim, Indianapolis, IN); Immobilon-P membrane (Millipore, Bedford, MA); Hyperfilm ECL, fluorescein-streptavidin, and Texas Red-streptavidin (Amersham Life Sciences, Arlington Heights, IL); Lumiglo (KPL); human TGF-β1, recombinant human TGF-β2 and -β3, recombinant human platelet-derived growth factor (PDGF)-BB, and anti-PDFG-BB (R & D Systems, Minneapolis, MN); Vectastain Elite ABC kit, avidin/biotin blocking kit, fluorescein anti-mouse IgG, Texas red anti-rabbit IgG, peroxidase-conjugated anti-goat IgG, biotinylated horse anti-mouse IgG, avidin-peroxidase, peroxidase-conjugated anti-mouse IgG, and 3,3′5,5′-tetramethylbenzidine (Vector Laboratories, Burlingame, CA); 3-amino-9-ethyl carbazol and mouse anti-human α-SMA (clone 1A4 ) (Sigma Chemical Co., St. Louis, MO); goat anti-human CD31/PECAM-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human von Willebrand factor (vWF) and mouse anti-human CD31/PECAM-1 (DAKO, Carpinteria, CA); polycarbonate PVP-F membranes (Neuro Probe, Inc, Gaithersburg, MD). .. Recombinant human bFGF was kindly provided by Scios Nova Inc., Mountain View, CA; soluble recombinant TGF-β type II receptor, prepared as described, was kindly provided by Philip Gotwals, Biogen, Cambridge, MA; SM1 antibody was kindly provided by Masanori Aikawa, Brigham and Women’s Hospital, Boston; rabbit anti-bovine CD31/PECAM-1 was kindly provided by Steven Albelda, University of Pennsylvania.

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