Structured Review

Boehringer Mannheim ptn r
Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( <t>pTN-R</t> ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p
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Images

1) Product Images from "Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection"

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.18-16-06218.1998

Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p
Figure Legend Snippet: Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p

Techniques Used: Staining, Incubation

2) Product Images from "Combined actions of Na+/K+-ATPase, NCX1 and glutamate dependent NMDA receptors in ischemic rat brain penumbra"

Article Title: Combined actions of Na+/K+-ATPase, NCX1 and glutamate dependent NMDA receptors in ischemic rat brain penumbra

Journal: Anatomy & Cell Biology

doi: 10.5115/acb.2010.43.3.201

Expression of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase monoclonal antibody (CNPase) in rats with pMCAO and in sham operated control rats. (A) Immunoblot was reacted with affinity purified anti-NeuN, anti-GFAP, and anti-CNPase antibodies, revealing 46-48, 50, and 46 KDa products. (B) Densitometric analysis revealed that focal cerebral ischemia produced a time-dependent decrease of NeuN (3 h: 52±6%; 6 h: 38±12%; 24 h: 29±8%, n=5, * P
Figure Legend Snippet: Expression of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase monoclonal antibody (CNPase) in rats with pMCAO and in sham operated control rats. (A) Immunoblot was reacted with affinity purified anti-NeuN, anti-GFAP, and anti-CNPase antibodies, revealing 46-48, 50, and 46 KDa products. (B) Densitometric analysis revealed that focal cerebral ischemia produced a time-dependent decrease of NeuN (3 h: 52±6%; 6 h: 38±12%; 24 h: 29±8%, n=5, * P

Techniques Used: Expressing, Affinity Purification, Produced

3) Product Images from "Persistent Neuroinflammatory Effects of Serial Exposure to Stress and Methamphetamine on the Blood-Brain Barrier"

Article Title: Persistent Neuroinflammatory Effects of Serial Exposure to Stress and Methamphetamine on the Blood-Brain Barrier

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

doi: 10.1007/s11481-012-9391-y

Markers of neuroinflammation, 7 days after treatment. Meth or saline was administered to previously stressed or control rats. GFAP and COX-2 protein expression was measured in cortex. a ) GFAP immunoreactivity was significantly increased by the combination
Figure Legend Snippet: Markers of neuroinflammation, 7 days after treatment. Meth or saline was administered to previously stressed or control rats. GFAP and COX-2 protein expression was measured in cortex. a ) GFAP immunoreactivity was significantly increased by the combination

Techniques Used: Expressing

Markers of neuroinflammation, 24 hrs after treatment. Meth or saline was administered to previously stressed or control rats. GFAP and COX-2 protein expression was measured in cortex. a ) GFAP immunoreactivity was not altered by stress or Meth. ( n =6 in
Figure Legend Snippet: Markers of neuroinflammation, 24 hrs after treatment. Meth or saline was administered to previously stressed or control rats. GFAP and COX-2 protein expression was measured in cortex. a ) GFAP immunoreactivity was not altered by stress or Meth. ( n =6 in

Techniques Used: Expressing

4) Product Images from "Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions"

Article Title: Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.23-13-05393.2003

Precipitation of Nogo-A from cell-surface biotinylated oligodendrocytes. Living cultures of oligodendrocytes were incubated with a cell-impermeable NHS-biotin analog. The biotinylated proteins were precipitated with streptavidin-coated beads, and the precipitate (Ppt) and supernatant (Sup) were analyzed by Western blot. All of the pellet and 1/10 of the supernatant were loaded. AS 472 showed the presence of Nogo-A in the precipitated sample, whereas the intracellular proteins β-tubulin and BiP were not present in the precipitated material. Intracellular Nogo-A, β–tubulin, and BiP were found in the supernatant.
Figure Legend Snippet: Precipitation of Nogo-A from cell-surface biotinylated oligodendrocytes. Living cultures of oligodendrocytes were incubated with a cell-impermeable NHS-biotin analog. The biotinylated proteins were precipitated with streptavidin-coated beads, and the precipitate (Ppt) and supernatant (Sup) were analyzed by Western blot. All of the pellet and 1/10 of the supernatant were loaded. AS 472 showed the presence of Nogo-A in the precipitated sample, whereas the intracellular proteins β-tubulin and BiP were not present in the precipitated material. Intracellular Nogo-A, β–tubulin, and BiP were found in the supernatant.

Techniques Used: Incubation, Western Blot

5) Product Images from "Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions"

Article Title: Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.23-13-05393.2003

Topology of Nogo-A in cultured oligodendrocytes. Oligodendrocytes (3–5 d in culture) were either fixed and completely permeabilized with Triton X-100 (Tx-100) or only plasma membrane permeabilized with digitonin (DIG). Cells were incubated with different α-Nogo-A antibodies. All α-Nogo-A Abs, mAb 11C7, AS Bianca, and AS 922 specifically recognize oligodendrocytes in dissociated rat optic nerve cultures (left row). After selective permeabilization with DIG, mAb 11C7, AS Bianca, and α-actin IgM mAb stain Nogo in the cytoplasm of oligodendrocytes (right row), whereas AS 922 does not. Antibodies against the luminal ER protein BiP were used as a control for the selective permeabilization. In DIG-permeabilized cells, no α-BiP staining could be detected, whereas all cells were strongly stained in the Tx-100-permeabilized cultures. Scale bar, 30 μm.
Figure Legend Snippet: Topology of Nogo-A in cultured oligodendrocytes. Oligodendrocytes (3–5 d in culture) were either fixed and completely permeabilized with Triton X-100 (Tx-100) or only plasma membrane permeabilized with digitonin (DIG). Cells were incubated with different α-Nogo-A antibodies. All α-Nogo-A Abs, mAb 11C7, AS Bianca, and AS 922 specifically recognize oligodendrocytes in dissociated rat optic nerve cultures (left row). After selective permeabilization with DIG, mAb 11C7, AS Bianca, and α-actin IgM mAb stain Nogo in the cytoplasm of oligodendrocytes (right row), whereas AS 922 does not. Antibodies against the luminal ER protein BiP were used as a control for the selective permeabilization. In DIG-permeabilized cells, no α-BiP staining could be detected, whereas all cells were strongly stained in the Tx-100-permeabilized cultures. Scale bar, 30 μm.

Techniques Used: Cell Culture, Incubation, Staining

6) Product Images from "The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast"

Article Title: The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast

Journal: Nucleic Acids Research

doi:

Association of Bfr1p with polyribosomes. ( A ) The top panel shows the OD 254 profile of a 15–45% sucrose gradient. Arrows indicate peaks representing small (40S) and large (60S) ribosomal subunits, and single ribosomes (80S). The center panel shows an α-HA western blot of gradient fractions concentrated and run on a 10% SDS–PAGE gel, to demonstrate the distribution of HA-Bfr1p in the gradient. The bottom panel shows an α-FLAG western blot of the same gradient fractions, demonstrating the distribution of FLAG-Scp160p. ( B ) Gradient profile and western blot analyses of a yeast lysate pre-treated with 30 mM EDTA. ( C ) Gradient profile and western blot analyses of a lysate pretreated with 50 U/ml RNase I.
Figure Legend Snippet: Association of Bfr1p with polyribosomes. ( A ) The top panel shows the OD 254 profile of a 15–45% sucrose gradient. Arrows indicate peaks representing small (40S) and large (60S) ribosomal subunits, and single ribosomes (80S). The center panel shows an α-HA western blot of gradient fractions concentrated and run on a 10% SDS–PAGE gel, to demonstrate the distribution of HA-Bfr1p in the gradient. The bottom panel shows an α-FLAG western blot of the same gradient fractions, demonstrating the distribution of FLAG-Scp160p. ( B ) Gradient profile and western blot analyses of a yeast lysate pre-treated with 30 mM EDTA. ( C ) Gradient profile and western blot analyses of a lysate pretreated with 50 U/ml RNase I.

Techniques Used: Western Blot, SDS Page

Deletion of BFR1 disrupts the association of Scp160p with cytosolic polyribosomes. ( A ) Sucrose gradient analysis of bfr1 null yeast expressing FLAG-Scp160p. ( B ) Distribution of HA-Bfr1p in an scp160 null strain. ( C ) Gel filtration chromatography followed by western blot analysis of an EDTA-treated soluble lysate from bfr1 null yeast. The solid arrow indicates FLAG-Scp160p. The open arrow indicates an ∼100 kDa cross-reacting endogenous yeast protein. ( D ) α-FLAG purification of FLAG-Scp160p from a bfr1 null strain, demonstrating continued association with Pab1p. ( E ) Subcellular fractionation of lysates from bfr1 null yeast demonstrating that a small amount of FLAG-Scp160p remains associated with the membrane pellet (solid arrow). As in (C), the open arrow indicates an endogenous yeast cross-reacting protein. ( F ) Subcellular fractionation of lysates from scp160 null yeast demonstrating that the membrane association of Bfr1p is apparently unaffected by loss of Scp160p.
Figure Legend Snippet: Deletion of BFR1 disrupts the association of Scp160p with cytosolic polyribosomes. ( A ) Sucrose gradient analysis of bfr1 null yeast expressing FLAG-Scp160p. ( B ) Distribution of HA-Bfr1p in an scp160 null strain. ( C ) Gel filtration chromatography followed by western blot analysis of an EDTA-treated soluble lysate from bfr1 null yeast. The solid arrow indicates FLAG-Scp160p. The open arrow indicates an ∼100 kDa cross-reacting endogenous yeast protein. ( D ) α-FLAG purification of FLAG-Scp160p from a bfr1 null strain, demonstrating continued association with Pab1p. ( E ) Subcellular fractionation of lysates from bfr1 null yeast demonstrating that a small amount of FLAG-Scp160p remains associated with the membrane pellet (solid arrow). As in (C), the open arrow indicates an endogenous yeast cross-reacting protein. ( F ) Subcellular fractionation of lysates from scp160 null yeast demonstrating that the membrane association of Bfr1p is apparently unaffected by loss of Scp160p.

Techniques Used: Expressing, Filtration, Chromatography, Western Blot, Purification, Fractionation

Bfr1p associates with mRNP complexes in the absence of Scp160p. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast expressing HA-Bfr1p in the presence (top panel) or absence (lower panel) of Scp160p. ( B ) α-HA purification and western blot analyses of S300 fractions (pooled 36–48 ml elution volume) derived from the same ΔSCP160 strain used in (A), probed to reveal both the HA-Bfr1p signal (top panel) and the Pab1p signal (lower panel). FT, flow-through fraction; E, elution fractions.
Figure Legend Snippet: Bfr1p associates with mRNP complexes in the absence of Scp160p. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast expressing HA-Bfr1p in the presence (top panel) or absence (lower panel) of Scp160p. ( B ) α-HA purification and western blot analyses of S300 fractions (pooled 36–48 ml elution volume) derived from the same ΔSCP160 strain used in (A), probed to reveal both the HA-Bfr1p signal (top panel) and the Pab1p signal (lower panel). FT, flow-through fraction; E, elution fractions.

Techniques Used: Filtration, Chromatography, Western Blot, Expressing, Purification, Derivative Assay, Flow Cytometry

Cytoplasmic localization and membrane association of Bfr1p. ( A ) Anti-GFP immunofluorescence microscopy of fixed cells expressing either GFP-Bfr1 or Scp160-GFP. Bfr1p (top panel) shows an almost identical distribution to Scp160p (bottom panel), appearing cytoplasmic with an enrichment around the nuclear envelope/ER. Panels to the right demonstrate DAPI staining of nuclear and mitochondrial DNA in the same cells. Arrows indicate clear examples of perinuclear staining. ( B ) Western blot analyses following subcellular fractionation of lysates from yeast expressing HA-Bfr1p and FLAG-Scp160p. Pellets were washed in the presence (right panels) or absence (left panels) of 50 U/ml RNase I. The open arrow indicates the location of an endogenous α-FLAG cross-reacting protein which does not associate with membrane pellets in this assay.
Figure Legend Snippet: Cytoplasmic localization and membrane association of Bfr1p. ( A ) Anti-GFP immunofluorescence microscopy of fixed cells expressing either GFP-Bfr1 or Scp160-GFP. Bfr1p (top panel) shows an almost identical distribution to Scp160p (bottom panel), appearing cytoplasmic with an enrichment around the nuclear envelope/ER. Panels to the right demonstrate DAPI staining of nuclear and mitochondrial DNA in the same cells. Arrows indicate clear examples of perinuclear staining. ( B ) Western blot analyses following subcellular fractionation of lysates from yeast expressing HA-Bfr1p and FLAG-Scp160p. Pellets were washed in the presence (right panels) or absence (left panels) of 50 U/ml RNase I. The open arrow indicates the location of an endogenous α-FLAG cross-reacting protein which does not associate with membrane pellets in this assay.

Techniques Used: Immunofluorescence, Microscopy, Expressing, Staining, Western Blot, Fractionation

RNA-dependent association of Bfr1p with Scp160p-containing mRNP complexes. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast co-expressing FLAG-Scp160p and HA-Bfr1p. ( B ) α-FLAG purification and western blot analyses of S300 void fractions derived from the same strain as in (A), with and without RNase pre-treatment. An α-Pab1p western blot of the same samples is shown as a control. FT, flow-through fraction; E, elution fractions.
Figure Legend Snippet: RNA-dependent association of Bfr1p with Scp160p-containing mRNP complexes. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast co-expressing FLAG-Scp160p and HA-Bfr1p. ( B ) α-FLAG purification and western blot analyses of S300 void fractions derived from the same strain as in (A), with and without RNase pre-treatment. An α-Pab1p western blot of the same samples is shown as a control. FT, flow-through fraction; E, elution fractions.

Techniques Used: Filtration, Chromatography, Western Blot, Expressing, Purification, Derivative Assay, Flow Cytometry

7) Product Images from "Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿"

Article Title: Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00415-06

Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P
Figure Legend Snippet: Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P

Techniques Used: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay

8) Product Images from "Migration Defects of cdk5−/− Neurons in the Developing Cerebellum is Cell Autonomous"

Article Title: Migration Defects of cdk5−/− Neurons in the Developing Cerebellum is Cell Autonomous

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.19-14-06017.1999

Cdk5 staining of granule cells in wild-type and chimeras visualized with confocal microscopy. Double immunostaining with antibodies β2/β3 GABA A ) and Cdk5. The cell surface localization of bd17 ( red ) surrounds the Cdk5-positive ( green ). Higher magnifications of boxed areas of A , C , and E are shown in B , D , and F , respectively. Granule cells are identified by membrane staining with bd17 and a diameter of 5–8 μm. The relative Cdk5 staining in granule cells is much lower than in Purkinjecells ( A , top left corner , and C , bottom right corner ). Granule cells of wild-type mice all contain Cdk5 ( A , B ). A few capillaries are stained by Cy3-conjugated anti-mouse IgG antibodies ( arrowheads in A ) and should not be confused with Cdk5-negative granule cells. The granule cell layer of the chimeric IGL contains both cdk5 +/+ and cdk5 −/− neurons ( C , D ). These can be seen at high magnification in D , with cdk5 +/+ granule cells indicated by the arrowheads and cdk5 −/− granule cells indicated by asterisks . The ectopic granule cell neurons of the chimeric molecular layer are Cdk5-deficient ( E , F ). Scale bar: A , C , E , 20 μm; B , D , F , 5 μm.
Figure Legend Snippet: Cdk5 staining of granule cells in wild-type and chimeras visualized with confocal microscopy. Double immunostaining with antibodies β2/β3 GABA A ) and Cdk5. The cell surface localization of bd17 ( red ) surrounds the Cdk5-positive ( green ). Higher magnifications of boxed areas of A , C , and E are shown in B , D , and F , respectively. Granule cells are identified by membrane staining with bd17 and a diameter of 5–8 μm. The relative Cdk5 staining in granule cells is much lower than in Purkinjecells ( A , top left corner , and C , bottom right corner ). Granule cells of wild-type mice all contain Cdk5 ( A , B ). A few capillaries are stained by Cy3-conjugated anti-mouse IgG antibodies ( arrowheads in A ) and should not be confused with Cdk5-negative granule cells. The granule cell layer of the chimeric IGL contains both cdk5 +/+ and cdk5 −/− neurons ( C , D ). These can be seen at high magnification in D , with cdk5 +/+ granule cells indicated by the arrowheads and cdk5 −/− granule cells indicated by asterisks . The ectopic granule cell neurons of the chimeric molecular layer are Cdk5-deficient ( E , F ). Scale bar: A , C , E , 20 μm; B , D , F , 5 μm.

Techniques Used: Staining, Confocal Microscopy, Double Immunostaining, Mouse Assay

9) Product Images from "Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection"

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.18-16-06218.1998

Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p
Figure Legend Snippet: Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p

Techniques Used: Staining, Incubation

10) Product Images from "Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection"

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.18-16-06218.1998

Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p
Figure Legend Snippet: Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p

Techniques Used: Staining, Incubation

11) Product Images from "Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿"

Article Title: Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00415-06

Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P
Figure Legend Snippet: Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P

Techniques Used: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay

12) Product Images from "Visual Experience Regulates Transient Expression and Dendritic Localization of Fragile X Mental Retardation Protein"

Article Title: Visual Experience Regulates Transient Expression and Dendritic Localization of Fragile X Mental Retardation Protein

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2185-04.2004

Visual experience induces a transient increase in FMRP levels in synaptoneurosome fractions from visual cortex. A , P60 DR rats were LE for 0, 30, or 60 min. Synaptoneurosome fractions were prepared from the visual cortices and probed for FMRP, NR1, and α-CaMKII expression by Western blot. In DR animals of this age (P60), FMRP levels peak at 30 min and return to baseline by 60 min. In contrast, α-CaMKII levels are elevated at 30 min and further increased at 60 min. Each lane shows synaptoneurosome-enriched fractions prepared from the visual cortex of an individual rat. B , The upregulation of FMRP induced by visual experience is NMDA receptor dependent. P45 dark-reared rats were exposed to light for the indicated times. The increased expression of FMRP observed at 15 min of light exposure is blocked by systemic administration of the NMDA receptor antagonist CPP (10 mg/kg). Systemic administration of MPEP (10-20 mg/kg) had no effect on FMRP expression (data not shown).
Figure Legend Snippet: Visual experience induces a transient increase in FMRP levels in synaptoneurosome fractions from visual cortex. A , P60 DR rats were LE for 0, 30, or 60 min. Synaptoneurosome fractions were prepared from the visual cortices and probed for FMRP, NR1, and α-CaMKII expression by Western blot. In DR animals of this age (P60), FMRP levels peak at 30 min and return to baseline by 60 min. In contrast, α-CaMKII levels are elevated at 30 min and further increased at 60 min. Each lane shows synaptoneurosome-enriched fractions prepared from the visual cortex of an individual rat. B , The upregulation of FMRP induced by visual experience is NMDA receptor dependent. P45 dark-reared rats were exposed to light for the indicated times. The increased expression of FMRP observed at 15 min of light exposure is blocked by systemic administration of the NMDA receptor antagonist CPP (10 mg/kg). Systemic administration of MPEP (10-20 mg/kg) had no effect on FMRP expression (data not shown).

Techniques Used: Expressing, Western Blot, Conditioned Place Preference

Antibody 2F5-1 is FMRP specific. A , Synaptoneurosome (SN) fractions were prepared from the cortex of wild-type (wt) and Fmr1 null mice (ko) and probed for FMRP, NR1, and α-CaMKII expression by Western blot. HeLa cell extracts are used as a positive control for FMRP expression. B , Sagittal sections from wild-type and Fmr1 knock-out mice were immunostained with FMRP or normal mouse IgG (MIgG; control). FMRP-positive cells are visible in the cerebral cortex and hippocampus of wild-type mice, but no FMRP-positive cells are detected in Fmr1 null mice. Scale bar, 10 μm.
Figure Legend Snippet: Antibody 2F5-1 is FMRP specific. A , Synaptoneurosome (SN) fractions were prepared from the cortex of wild-type (wt) and Fmr1 null mice (ko) and probed for FMRP, NR1, and α-CaMKII expression by Western blot. HeLa cell extracts are used as a positive control for FMRP expression. B , Sagittal sections from wild-type and Fmr1 knock-out mice were immunostained with FMRP or normal mouse IgG (MIgG; control). FMRP-positive cells are visible in the cerebral cortex and hippocampus of wild-type mice, but no FMRP-positive cells are detected in Fmr1 null mice. Scale bar, 10 μm.

Techniques Used: Mouse Assay, Expressing, Western Blot, Positive Control, Knock-Out

13) Product Images from "Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase"

Article Title: Lipopolysaccharide-Induced Activation of ?2-Integrin Function in Macrophages Requires Irak Kinase Activity, p38 Mitogen- Activated Protein Kinase, and the Rap1 GTPase

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.2.438-448.2001

(A) LPS-induced spreading requires Irak and MyD88; Irak-induced spreading is dependent on p38 and Rap1. Shown here is the induction of macrophage spreading by Irak and MyD88 constructs in the presence or absence of LPS, SB202190, or TcdB-1470. Macrophages were microinjected with FITC-dextran (−) or cDNAs encoding for myc-tagged Irak wild-type and mutant constructs or AU1-tagged MyD88▵. Cells were returned to the incubator for expression of the constructs and then incubated as indicated either with LPS (1 μg/ml) for 10 min, SB202190 (1 μM) for 20 min, or TcdB-1470 (10 pg/ml) for 2 h. Cells were then fixed, stained with anti-myc or anti-AU1 antibodies and rhodamine-phalloidin, and assayed for spreading. (B) Irak induces spreading in J774.A1 macrophages. Macrophages were microinjected with FITC-dextran (control) or a cDNA construct encoding for myc-tagged Irak. Cells were returned to the incubator for expression of the construct, fixed and stained with anti-myc and rhodamine-phalloidin, and assayed for spreading.
Figure Legend Snippet: (A) LPS-induced spreading requires Irak and MyD88; Irak-induced spreading is dependent on p38 and Rap1. Shown here is the induction of macrophage spreading by Irak and MyD88 constructs in the presence or absence of LPS, SB202190, or TcdB-1470. Macrophages were microinjected with FITC-dextran (−) or cDNAs encoding for myc-tagged Irak wild-type and mutant constructs or AU1-tagged MyD88▵. Cells were returned to the incubator for expression of the constructs and then incubated as indicated either with LPS (1 μg/ml) for 10 min, SB202190 (1 μM) for 20 min, or TcdB-1470 (10 pg/ml) for 2 h. Cells were then fixed, stained with anti-myc or anti-AU1 antibodies and rhodamine-phalloidin, and assayed for spreading. (B) Irak induces spreading in J774.A1 macrophages. Macrophages were microinjected with FITC-dextran (control) or a cDNA construct encoding for myc-tagged Irak. Cells were returned to the incubator for expression of the construct, fixed and stained with anti-myc and rhodamine-phalloidin, and assayed for spreading.

Techniques Used: Construct, Mutagenesis, Expressing, Incubation, Staining

14) Product Images from "Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast"

Article Title: Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast

Journal: Nucleic Acids Research

doi:

Characterization of the 450 kDa Scp160p species. ( A ) α-FLAG western of FLAG–His6–Scp160p expressed and purified from Pichia , and then run on the S-300 gel filtration column. ( B ) Twelve microliters of purified FLAG–His6–Scp160p was run on SDS–PAGE and stained with colloidal G250 Coomassie. As shown, no other proteins were detected in stoichiometric quantities with Scp160p. ( C ) Top, α-HA western blot of purified extract from yeast co-expressing both FLAG–Scp160p and HA–Scp160p. No HA–Scp160p appears to co-isolate during the α-FLAG purification. Bottom, α-FLAG western blot of the same samples showing that FLAG–Scp160p was successfully isolated.
Figure Legend Snippet: Characterization of the 450 kDa Scp160p species. ( A ) α-FLAG western of FLAG–His6–Scp160p expressed and purified from Pichia , and then run on the S-300 gel filtration column. ( B ) Twelve microliters of purified FLAG–His6–Scp160p was run on SDS–PAGE and stained with colloidal G250 Coomassie. As shown, no other proteins were detected in stoichiometric quantities with Scp160p. ( C ) Top, α-HA western blot of purified extract from yeast co-expressing both FLAG–Scp160p and HA–Scp160p. No HA–Scp160p appears to co-isolate during the α-FLAG purification. Bottom, α-FLAG western blot of the same samples showing that FLAG–Scp160p was successfully isolated.

Techniques Used: Western Blot, Purification, Filtration, SDS Page, Staining, Expressing, Isolation

Gel-filtration analysis of Scp160p-containing complexes. Cell lysates from yeast expressing FLAG–Scp160p were passed over a 120 ml S-300 Sephacryl column, and 2 ml fractions [except (A) where fractions were 4 ml] collected between 35 and 70 ml were assayed by α-FLAG western. Solid arrows indicate the position of the 160 kDa Scp160p band, the open arrows indicate the 100 kDa endogenous cross-reacting band. ( A ) No treatment, showing presence of FLAG–Scp160p in the void fractions. ( B ) Treatment with 30 mM EDTA, showing retention of Scp160p in the void fractions. ( C ) Treatment with 50 U/ml RNase for 10 min at room temperature, partially releasing a ~450 kDa complex. ( D ) Thirty minute RNase treatment, showing only the 450 kDa complex. ( E ) Treatment with 75 mM NaCl ( F ). Treatment with 150 mM NaCl. ( G ) Treatment with 1 M NaCl.
Figure Legend Snippet: Gel-filtration analysis of Scp160p-containing complexes. Cell lysates from yeast expressing FLAG–Scp160p were passed over a 120 ml S-300 Sephacryl column, and 2 ml fractions [except (A) where fractions were 4 ml] collected between 35 and 70 ml were assayed by α-FLAG western. Solid arrows indicate the position of the 160 kDa Scp160p band, the open arrows indicate the 100 kDa endogenous cross-reacting band. ( A ) No treatment, showing presence of FLAG–Scp160p in the void fractions. ( B ) Treatment with 30 mM EDTA, showing retention of Scp160p in the void fractions. ( C ) Treatment with 50 U/ml RNase for 10 min at room temperature, partially releasing a ~450 kDa complex. ( D ) Thirty minute RNase treatment, showing only the 450 kDa complex. ( E ) Treatment with 75 mM NaCl ( F ). Treatment with 150 mM NaCl. ( G ) Treatment with 1 M NaCl.

Techniques Used: Filtration, Expressing, Western Blot

α-FLAG affinity purification of Scp160p-containing complexes. Cell lysates expressing the indicated alleles of Scp160p were first passed over an S-300 gel filtration column, followed by an α-FLAG immunoaffinity column as described in the Materials and Methods section. Lanes: crude, starting material loaded onto S-300 column; void, pooled fractions containing void volume collected after S-300 run; FT, flow-through material after two passes over α-FLAG column; E1-4, 1 ml elution fractions collected after adding FLAG peptide. ( A ) Top, α-FLAG western blot, showing that FLAG–Scp160p appears predominantly in the first several fractions following treatment with elution buffer. Bottom, colloidal G250 Coomassie stained gel of concentrated samples. ( B ) α-Pab1p western, demonstrating co-isolation of Pab1p with Scp160p. ( C ) RNase treatment prior to α-FLAG purification disrupts the Pab1p interaction. Upper panel, α-FLAG western; lower panel, α-Pab1p western. ( D ) α-Pub1p western, demonstrates that this protein does not co-purify with Scp160p. ( E ) Negative control showing colloidal G250 Coomassie stained gel of fractions from a wild-type (non-FLAG–Scp160p) purification. ( F ) α-Pab1p western of negative control fractions, showing no detectable Pab1p signal. ( G ) Background levels of Pab1p non-specifically isolated during α-FLAG purification from yeast expressing an unrelated protein, FLAG–GALT. Since FLAG–GALT exists primarily as an 88 kDa dimer in vivo , lysates were not pre-purified over the S-300 column.
Figure Legend Snippet: α-FLAG affinity purification of Scp160p-containing complexes. Cell lysates expressing the indicated alleles of Scp160p were first passed over an S-300 gel filtration column, followed by an α-FLAG immunoaffinity column as described in the Materials and Methods section. Lanes: crude, starting material loaded onto S-300 column; void, pooled fractions containing void volume collected after S-300 run; FT, flow-through material after two passes over α-FLAG column; E1-4, 1 ml elution fractions collected after adding FLAG peptide. ( A ) Top, α-FLAG western blot, showing that FLAG–Scp160p appears predominantly in the first several fractions following treatment with elution buffer. Bottom, colloidal G250 Coomassie stained gel of concentrated samples. ( B ) α-Pab1p western, demonstrating co-isolation of Pab1p with Scp160p. ( C ) RNase treatment prior to α-FLAG purification disrupts the Pab1p interaction. Upper panel, α-FLAG western; lower panel, α-Pab1p western. ( D ) α-Pub1p western, demonstrates that this protein does not co-purify with Scp160p. ( E ) Negative control showing colloidal G250 Coomassie stained gel of fractions from a wild-type (non-FLAG–Scp160p) purification. ( F ) α-Pab1p western of negative control fractions, showing no detectable Pab1p signal. ( G ) Background levels of Pab1p non-specifically isolated during α-FLAG purification from yeast expressing an unrelated protein, FLAG–GALT. Since FLAG–GALT exists primarily as an 88 kDa dimer in vivo , lysates were not pre-purified over the S-300 column.

Techniques Used: Affinity Purification, Expressing, Filtration, Flow Cytometry, Western Blot, Staining, Isolation, Purification, Negative Control, In Vivo

Epitope-tagged and deletion alleles of Scp160p expressed in yeast. ( A ) Diagram of FLAG-tagged and deletion alleles of SCP160 substituted into the yJJ52 haploid genome in place of the endogenous SCP160 allele. Replacement of the endogenous allele in both cases was confirmed by PCR of isolated genomic DNA as described in Materials and Methods. ( B ) Western blot analysis using anti-FLAG monoclonal antibody M2. Lane 1, cellular extract from normal yJJ52. Lane 2, an extract from yJJ52 cells modified to encode FLAG–Scp160p in place of their endogenous Scp160p (JFy1511). The solid arrow indicates the position of the ~160 kDa Scp160p band; the open arrow indicates an abundant, anti-FLAG cross-reacting yeast protein of unknown identity that is used as an internal control in several experiments.
Figure Legend Snippet: Epitope-tagged and deletion alleles of Scp160p expressed in yeast. ( A ) Diagram of FLAG-tagged and deletion alleles of SCP160 substituted into the yJJ52 haploid genome in place of the endogenous SCP160 allele. Replacement of the endogenous allele in both cases was confirmed by PCR of isolated genomic DNA as described in Materials and Methods. ( B ) Western blot analysis using anti-FLAG monoclonal antibody M2. Lane 1, cellular extract from normal yJJ52. Lane 2, an extract from yJJ52 cells modified to encode FLAG–Scp160p in place of their endogenous Scp160p (JFy1511). The solid arrow indicates the position of the ~160 kDa Scp160p band; the open arrow indicates an abundant, anti-FLAG cross-reacting yeast protein of unknown identity that is used as an internal control in several experiments.

Techniques Used: Polymerase Chain Reaction, Isolation, Western Blot, Modification

Bfr1 associates with Scp160p complexes. Yeast expressing either FLAG–Scp160p and HA–Bfr1p together, or HA–Bfr1p in the presence of native, untagged Scp160p, were lysed and subjected to the purification protocol described in Figure 4. Top, α-FLAG western blot. Center, α-HA western blot showing co-elution of HA–Bfr1. Bottom, α-HA western blot showing the absence of HA–Bfr1p in elutions from lysates lacking FLAG–Scp160p.
Figure Legend Snippet: Bfr1 associates with Scp160p complexes. Yeast expressing either FLAG–Scp160p and HA–Bfr1p together, or HA–Bfr1p in the presence of native, untagged Scp160p, were lysed and subjected to the purification protocol described in Figure 4. Top, α-FLAG western blot. Center, α-HA western blot showing co-elution of HA–Bfr1. Bottom, α-HA western blot showing the absence of HA–Bfr1p in elutions from lysates lacking FLAG–Scp160p.

Techniques Used: Expressing, Purification, Western Blot, Co-Elution Assay

Scp160p associates with polyribosomes. ( A ) Top panel shows OD 254 profile of the 15–45% sucrose gradient. Arrows indicate peaks representing small (40S) and large (60S) ribosomal subunits, single ribosomes (80S) and polyribosomes ( > 80S). Bottom panel shows an α-FLAG western blot of gradient fractions concentrated and run on a 10% SDS–PAGE gel. Position of the 160 kDa band is indicated (solid arrow), as well as the non-specific. 100 kDa band which serves as an internal control (open arrow). ( B ) Gradient profile and western blot analysis of a sample pre-treated with 30 mM EDTA. ( C ) Gradient profile and western blot analysis of a sample pretreated with 50 U/ml RNase One.
Figure Legend Snippet: Scp160p associates with polyribosomes. ( A ) Top panel shows OD 254 profile of the 15–45% sucrose gradient. Arrows indicate peaks representing small (40S) and large (60S) ribosomal subunits, single ribosomes (80S) and polyribosomes ( > 80S). Bottom panel shows an α-FLAG western blot of gradient fractions concentrated and run on a 10% SDS–PAGE gel. Position of the 160 kDa band is indicated (solid arrow), as well as the non-specific. 100 kDa band which serves as an internal control (open arrow). ( B ) Gradient profile and western blot analysis of a sample pre-treated with 30 mM EDTA. ( C ) Gradient profile and western blot analysis of a sample pretreated with 50 U/ml RNase One.

Techniques Used: Western Blot, SDS Page

Schematic representation of the FLAG–Scp160p purification protocol.
Figure Legend Snippet: Schematic representation of the FLAG–Scp160p purification protocol.

Techniques Used: Purification

15) Product Images from "Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection"

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.18-16-06218.1998

Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p
Figure Legend Snippet: Effect of TN-R on microglial adhesion to PLL. A , Adhesion pattern of microglial cells on BSA-containing ( PLL + BSA ), TN-R-containing ( PLL + TN-R ), and LN-containing ( PLL + LN ) PLL substrates after 30 min ( a–c ) and 24 hr ( d–f ) in culture. a–c , Cells were stained with toluidine blue. Scale bar, 75 μm. B , Effect of monoclonal ( tn-R1 , tn-R2 , and tn-R6 ) and polyclonal ( pTN-R ) Abs to TN-R on microglial adhesion to PLL + BSA and PLL + TN-R substrates. Protein substrates were incubated in the absence ( -Ab ) or presence of Abs to TN-R before plating the cells. The number of adherent cells after 30 min of incubation on PLL + BSA (control substrates) was set as 100%. Values represent the mean ± SD of one representative of three to five independent experiments performed in triplicate. Asterisks indicate statistically significant differences in the number of adherent cells. * p

Techniques Used: Staining, Incubation

16) Product Images from "Characterization of a Novel Trypanosome Lytic Factor from Human Serum"

Article Title: Characterization of a Novel Trypanosome Lytic Factor from Human Serum

Journal: Infection and Immunity

doi:

TLF2 is a protein complex. One microgram of TLF2 was immunoprecipitated by antibodies to human Hp (MAb; α-Hp), human apoA-I (polyclonal antibodies from sheep and goat; α-apoA-I), and human IgM (MAb; α-IgM). Following immunoprecipitation, the pellet (P) and supernatant (S) were separated by reducing SDS-PAGE (12% gel) and transferred to PVDF membranes. The IgM μ chain was detected with rabbit anti-IgM μ-chain antibody followed by goat anti-rabbit IgG-HRP and exposed for 30 s by ECL.
Figure Legend Snippet: TLF2 is a protein complex. One microgram of TLF2 was immunoprecipitated by antibodies to human Hp (MAb; α-Hp), human apoA-I (polyclonal antibodies from sheep and goat; α-apoA-I), and human IgM (MAb; α-IgM). Following immunoprecipitation, the pellet (P) and supernatant (S) were separated by reducing SDS-PAGE (12% gel) and transferred to PVDF membranes. The IgM μ chain was detected with rabbit anti-IgM μ-chain antibody followed by goat anti-rabbit IgG-HRP and exposed for 30 s by ECL.

Techniques Used: Immunoprecipitation, SDS Page

17) Product Images from "Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿"

Article Title: Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00415-06

Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P
Figure Legend Snippet: Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P

Techniques Used: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay

18) Product Images from "Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿"

Article Title: Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00415-06

Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P
Figure Legend Snippet: Specific IgG isotype responses in mice immunized with rAd97c. Mice (five per group) were immunized with rAdP97c or with rAdGFP (negative control). Pools of sera (A) and BALs (B) collected at day 60 p.i. were examined for P97c-specific IgG isotypes by ELISA in duplicate. The data represent the mean OD 405 plus the standard deviations. *, significant difference ( P

Techniques Used: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay

19) Product Images from "Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast"

Article Title: Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast

Journal: Nucleic Acids Research

doi:

Bfr1 associates with Scp160p complexes. Yeast expressing either FLAG–Scp160p and HA–Bfr1p together, or HA–Bfr1p in the presence of native, untagged Scp160p, were lysed and subjected to the purification protocol described in Figure 4. Top, α-FLAG western blot. Center, α-HA western blot showing co-elution of HA–Bfr1. Bottom, α-HA western blot showing the absence of HA–Bfr1p in elutions from lysates lacking FLAG–Scp160p.
Figure Legend Snippet: Bfr1 associates with Scp160p complexes. Yeast expressing either FLAG–Scp160p and HA–Bfr1p together, or HA–Bfr1p in the presence of native, untagged Scp160p, were lysed and subjected to the purification protocol described in Figure 4. Top, α-FLAG western blot. Center, α-HA western blot showing co-elution of HA–Bfr1. Bottom, α-HA western blot showing the absence of HA–Bfr1p in elutions from lysates lacking FLAG–Scp160p.

Techniques Used: Expressing, Purification, Western Blot, Co-Elution Assay

20) Product Images from "Visual Experience Regulates Transient Expression and Dendritic Localization of Fragile X Mental Retardation Protein"

Article Title: Visual Experience Regulates Transient Expression and Dendritic Localization of Fragile X Mental Retardation Protein

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2185-04.2004

Visual experience induces a transient increase in FMRP levels in synaptoneurosome fractions from visual cortex. A , P60 DR rats were LE for 0, 30, or 60 min. Synaptoneurosome fractions were prepared from the visual cortices and probed for FMRP, NR1, and α-CaMKII expression by Western blot. In DR animals of this age (P60), FMRP levels peak at 30 min and return to baseline by 60 min. In contrast, α-CaMKII levels are elevated at 30 min and further increased at 60 min. Each lane shows synaptoneurosome-enriched fractions prepared from the visual cortex of an individual rat. B , The upregulation of FMRP induced by visual experience is NMDA receptor dependent. P45 dark-reared rats were exposed to light for the indicated times. The increased expression of FMRP observed at 15 min of light exposure is blocked by systemic administration of the NMDA receptor antagonist CPP (10 mg/kg). Systemic administration of MPEP (10-20 mg/kg) had no effect on FMRP expression (data not shown).
Figure Legend Snippet: Visual experience induces a transient increase in FMRP levels in synaptoneurosome fractions from visual cortex. A , P60 DR rats were LE for 0, 30, or 60 min. Synaptoneurosome fractions were prepared from the visual cortices and probed for FMRP, NR1, and α-CaMKII expression by Western blot. In DR animals of this age (P60), FMRP levels peak at 30 min and return to baseline by 60 min. In contrast, α-CaMKII levels are elevated at 30 min and further increased at 60 min. Each lane shows synaptoneurosome-enriched fractions prepared from the visual cortex of an individual rat. B , The upregulation of FMRP induced by visual experience is NMDA receptor dependent. P45 dark-reared rats were exposed to light for the indicated times. The increased expression of FMRP observed at 15 min of light exposure is blocked by systemic administration of the NMDA receptor antagonist CPP (10 mg/kg). Systemic administration of MPEP (10-20 mg/kg) had no effect on FMRP expression (data not shown).

Techniques Used: Expressing, Western Blot, Conditioned Place Preference

Antibody 2F5-1 is FMRP specific. A , Synaptoneurosome (SN) fractions were prepared from the cortex of wild-type (wt) and Fmr1 null mice (ko) and probed for FMRP, NR1, and α-CaMKII expression by Western blot. HeLa cell extracts are used as a positive control for FMRP expression. B , Sagittal sections from wild-type and Fmr1 knock-out mice were immunostained with FMRP or normal mouse IgG (MIgG; control). FMRP-positive cells are visible in the cerebral cortex and hippocampus of wild-type mice, but no FMRP-positive cells are detected in Fmr1 null mice. Scale bar, 10 μm.
Figure Legend Snippet: Antibody 2F5-1 is FMRP specific. A , Synaptoneurosome (SN) fractions were prepared from the cortex of wild-type (wt) and Fmr1 null mice (ko) and probed for FMRP, NR1, and α-CaMKII expression by Western blot. HeLa cell extracts are used as a positive control for FMRP expression. B , Sagittal sections from wild-type and Fmr1 knock-out mice were immunostained with FMRP or normal mouse IgG (MIgG; control). FMRP-positive cells are visible in the cerebral cortex and hippocampus of wild-type mice, but no FMRP-positive cells are detected in Fmr1 null mice. Scale bar, 10 μm.

Techniques Used: Mouse Assay, Expressing, Western Blot, Positive Control, Knock-Out

21) Product Images from "ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores"

Article Title: ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201408089

ARHGEF17 fragment restores SAC function independently of catalytic activity for Rho GEF of ARHGEF17. (A) Schematic depiction of hARHGEF17 variants used in phenotypic rescue assays: FL: hARHGEF17 (1–2,063)-mEGFP; FL Y1216A: hARHGEF17 (1–2,063)-Y1216A-mEGFP; ΔN: hARHGEF17 (667–2,063)-mEGFP; ΔNC: hARHGEF17 (667–1,306)-mEGFP; ΔNC Y1216A: hARHGEF17 (667–1,306)-Y1216A-mEGFP; ΔC-siRa: hARHGEF17 (1–582)-mEGFP; ΔC-siRb: hARHGEF17 (109–664)-mEGFP; ΔN1: mEGFP-hARHGEF17 (1,304–2,063)-mEGFP; and ΔN2: hARHGEF17 (1,304–2,063)-mEGFP. Gray boxes indicate GEF activity domain (Dbl-homologous domain); Y1216A indicates the inactivating mutation in the GEF domain; red lines indicate sites for mutations for siRNA resistance. (B) Immunoblot analysis of hARHGEF17 fragments fused to GFP (detected with anti-GFP). GAPDH, loading control. (C and D) Rescue of hARHGEF17 knockdown-induced polylobulation (C) or SAC defect (D). Comparison of polylobed (C; normalized to Scrambled) or prometaphase population (D; nocodazole treated) rescued with hARHGEF17 fragments (ΔN-mEGFP, ΔNC-mEGFP, and ΔNC Y1216A-mEGFP) of > 11,500 (C) or > 6,300 (D) cells/three independent experiments. (E) Phenotypic analysis of cytokinesis defects (binucleation) of a catalytically inactive mutant of full-length ARHGEF17 (FL Y1216A) during mitosis. (left) H2B-mCherry, wild-type full-length (FL-mEGFP), catalytically inactive (FL Y1216A-mEGFP), or cells 24 h after CT04 treatment (0.25 mg/ml; FL + CT04). (right) Comparison of binuclear population of > 3,000 cells/three independent experiments. Bar graphs show mean ± SD. **, P
Figure Legend Snippet: ARHGEF17 fragment restores SAC function independently of catalytic activity for Rho GEF of ARHGEF17. (A) Schematic depiction of hARHGEF17 variants used in phenotypic rescue assays: FL: hARHGEF17 (1–2,063)-mEGFP; FL Y1216A: hARHGEF17 (1–2,063)-Y1216A-mEGFP; ΔN: hARHGEF17 (667–2,063)-mEGFP; ΔNC: hARHGEF17 (667–1,306)-mEGFP; ΔNC Y1216A: hARHGEF17 (667–1,306)-Y1216A-mEGFP; ΔC-siRa: hARHGEF17 (1–582)-mEGFP; ΔC-siRb: hARHGEF17 (109–664)-mEGFP; ΔN1: mEGFP-hARHGEF17 (1,304–2,063)-mEGFP; and ΔN2: hARHGEF17 (1,304–2,063)-mEGFP. Gray boxes indicate GEF activity domain (Dbl-homologous domain); Y1216A indicates the inactivating mutation in the GEF domain; red lines indicate sites for mutations for siRNA resistance. (B) Immunoblot analysis of hARHGEF17 fragments fused to GFP (detected with anti-GFP). GAPDH, loading control. (C and D) Rescue of hARHGEF17 knockdown-induced polylobulation (C) or SAC defect (D). Comparison of polylobed (C; normalized to Scrambled) or prometaphase population (D; nocodazole treated) rescued with hARHGEF17 fragments (ΔN-mEGFP, ΔNC-mEGFP, and ΔNC Y1216A-mEGFP) of > 11,500 (C) or > 6,300 (D) cells/three independent experiments. (E) Phenotypic analysis of cytokinesis defects (binucleation) of a catalytically inactive mutant of full-length ARHGEF17 (FL Y1216A) during mitosis. (left) H2B-mCherry, wild-type full-length (FL-mEGFP), catalytically inactive (FL Y1216A-mEGFP), or cells 24 h after CT04 treatment (0.25 mg/ml; FL + CT04). (right) Comparison of binuclear population of > 3,000 cells/three independent experiments. Bar graphs show mean ± SD. **, P

Techniques Used: Activity Assay, Mutagenesis

ARHGEF17 and Mps1 interact during mitosis. (A) Coimmunoprecipitation of ARHGEF17 with Mps1: LAP-tagged Mps1 (LAP-Mps1) and mCherry-tagged ARHGEF17 (ARHGEF17-mCherry) were immunoprecipitated using GFP-binding protein coupled to agarose beads. Input, supernatants (Unbound), and immunoprecipitates (Bound) were analyzed by Western Blot (anti-GFP and anti-mCherry). (B and C) FCCS of Mps1 and ARHGEF17. Exemplary cells (B; yellow crosses mark position for FCCS measurement) and normalized cross-correlation (C) of ARHGEF17-mCherry and LAP-Mps1 with or without reversine treatment of > 40 cells (specific numbers indicated)/three independent experiments. (D) Coimmunoprecipitation of ARHGEF17 fragments with Mps1: LAP-Mps1 and mCherry-tagged ARHGEF17 fragments (ΔNC-mCherry and ΔNC Y1216A-mCherry) were precipitated using GFP-binding protein coupled to agarose beads. Input and precipitates were analyzed by Western blot (anti-GFP and anti-mCherry). (E and F) FCCS of Mps1 and ARHGEF17 fragments. Exemplary cells (E) and normalized cross-correlation (F) of LAP-Mps1 and ARHGEF17 fragments (ΔNC-mCherry and ΔNC Y1216A-mCherry) of > 40 cells (specific numbers indicated)/three independent experiments. (G) In vitro pull-down of ARHGEF17 fragments with Mps1. His-tagged Mps1 (bait) and untagged ARHGEF17 fragments (ΔNC; target) were precipitated using His-tag binding protein coupled to Talon beads. Input, supernatants (Unbound), and precipitates (Bound) were analyzed by Western blot (anti–His-tag [middle] and anti-ARHGEF17 [bottom]). Coomassie brilliant blue staining was used as internal protein control in each condition. Boxes show median, 25–75%; whiskers show 1.5× interquartile range. **, P
Figure Legend Snippet: ARHGEF17 and Mps1 interact during mitosis. (A) Coimmunoprecipitation of ARHGEF17 with Mps1: LAP-tagged Mps1 (LAP-Mps1) and mCherry-tagged ARHGEF17 (ARHGEF17-mCherry) were immunoprecipitated using GFP-binding protein coupled to agarose beads. Input, supernatants (Unbound), and immunoprecipitates (Bound) were analyzed by Western Blot (anti-GFP and anti-mCherry). (B and C) FCCS of Mps1 and ARHGEF17. Exemplary cells (B; yellow crosses mark position for FCCS measurement) and normalized cross-correlation (C) of ARHGEF17-mCherry and LAP-Mps1 with or without reversine treatment of > 40 cells (specific numbers indicated)/three independent experiments. (D) Coimmunoprecipitation of ARHGEF17 fragments with Mps1: LAP-Mps1 and mCherry-tagged ARHGEF17 fragments (ΔNC-mCherry and ΔNC Y1216A-mCherry) were precipitated using GFP-binding protein coupled to agarose beads. Input and precipitates were analyzed by Western blot (anti-GFP and anti-mCherry). (E and F) FCCS of Mps1 and ARHGEF17 fragments. Exemplary cells (E) and normalized cross-correlation (F) of LAP-Mps1 and ARHGEF17 fragments (ΔNC-mCherry and ΔNC Y1216A-mCherry) of > 40 cells (specific numbers indicated)/three independent experiments. (G) In vitro pull-down of ARHGEF17 fragments with Mps1. His-tagged Mps1 (bait) and untagged ARHGEF17 fragments (ΔNC; target) were precipitated using His-tag binding protein coupled to Talon beads. Input, supernatants (Unbound), and precipitates (Bound) were analyzed by Western blot (anti–His-tag [middle] and anti-ARHGEF17 [bottom]). Coomassie brilliant blue staining was used as internal protein control in each condition. Boxes show median, 25–75%; whiskers show 1.5× interquartile range. **, P

Techniques Used: Immunoprecipitation, Binding Assay, Western Blot, In Vitro, Staining

22) Product Images from "The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast"

Article Title: The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast

Journal: Nucleic Acids Research

doi:

Association of Bfr1p with polyribosomes. ( A ) The top panel shows the OD 254 profile of a 15–45% sucrose gradient. Arrows indicate peaks representing small (40S) and large (60S) ribosomal subunits, and single ribosomes (80S). The center panel shows an α-HA western blot of gradient fractions concentrated and run on a 10% SDS–PAGE gel, to demonstrate the distribution of HA-Bfr1p in the gradient. The bottom panel shows an α-FLAG western blot of the same gradient fractions, demonstrating the distribution of FLAG-Scp160p. ( B ) Gradient profile and western blot analyses of a yeast lysate pre-treated with 30 mM EDTA. ( C ) Gradient profile and western blot analyses of a lysate pretreated with 50 U/ml RNase I.
Figure Legend Snippet: Association of Bfr1p with polyribosomes. ( A ) The top panel shows the OD 254 profile of a 15–45% sucrose gradient. Arrows indicate peaks representing small (40S) and large (60S) ribosomal subunits, and single ribosomes (80S). The center panel shows an α-HA western blot of gradient fractions concentrated and run on a 10% SDS–PAGE gel, to demonstrate the distribution of HA-Bfr1p in the gradient. The bottom panel shows an α-FLAG western blot of the same gradient fractions, demonstrating the distribution of FLAG-Scp160p. ( B ) Gradient profile and western blot analyses of a yeast lysate pre-treated with 30 mM EDTA. ( C ) Gradient profile and western blot analyses of a lysate pretreated with 50 U/ml RNase I.

Techniques Used: Western Blot, SDS Page

Deletion of BFR1 disrupts the association of Scp160p with cytosolic polyribosomes. ( A ) Sucrose gradient analysis of bfr1 null yeast expressing FLAG-Scp160p. ( B ) Distribution of HA-Bfr1p in an scp160 null strain. ( C ) Gel filtration chromatography followed by western blot analysis of an EDTA-treated soluble lysate from bfr1 null yeast. The solid arrow indicates FLAG-Scp160p. The open arrow indicates an ∼100 kDa cross-reacting endogenous yeast protein. ( D ) α-FLAG purification of FLAG-Scp160p from a bfr1 null strain, demonstrating continued association with Pab1p. ( E ) Subcellular fractionation of lysates from bfr1 null yeast demonstrating that a small amount of FLAG-Scp160p remains associated with the membrane pellet (solid arrow). As in (C), the open arrow indicates an endogenous yeast cross-reacting protein. ( F ) Subcellular fractionation of lysates from scp160 null yeast demonstrating that the membrane association of Bfr1p is apparently unaffected by loss of Scp160p.
Figure Legend Snippet: Deletion of BFR1 disrupts the association of Scp160p with cytosolic polyribosomes. ( A ) Sucrose gradient analysis of bfr1 null yeast expressing FLAG-Scp160p. ( B ) Distribution of HA-Bfr1p in an scp160 null strain. ( C ) Gel filtration chromatography followed by western blot analysis of an EDTA-treated soluble lysate from bfr1 null yeast. The solid arrow indicates FLAG-Scp160p. The open arrow indicates an ∼100 kDa cross-reacting endogenous yeast protein. ( D ) α-FLAG purification of FLAG-Scp160p from a bfr1 null strain, demonstrating continued association with Pab1p. ( E ) Subcellular fractionation of lysates from bfr1 null yeast demonstrating that a small amount of FLAG-Scp160p remains associated with the membrane pellet (solid arrow). As in (C), the open arrow indicates an endogenous yeast cross-reacting protein. ( F ) Subcellular fractionation of lysates from scp160 null yeast demonstrating that the membrane association of Bfr1p is apparently unaffected by loss of Scp160p.

Techniques Used: Expressing, Filtration, Chromatography, Western Blot, Purification, Fractionation

Bfr1p associates with mRNP complexes in the absence of Scp160p. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast expressing HA-Bfr1p in the presence (top panel) or absence (lower panel) of Scp160p. ( B ) α-HA purification and western blot analyses of S300 fractions (pooled 36–48 ml elution volume) derived from the same ΔSCP160 strain used in (A), probed to reveal both the HA-Bfr1p signal (top panel) and the Pab1p signal (lower panel). FT, flow-through fraction; E, elution fractions.
Figure Legend Snippet: Bfr1p associates with mRNP complexes in the absence of Scp160p. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast expressing HA-Bfr1p in the presence (top panel) or absence (lower panel) of Scp160p. ( B ) α-HA purification and western blot analyses of S300 fractions (pooled 36–48 ml elution volume) derived from the same ΔSCP160 strain used in (A), probed to reveal both the HA-Bfr1p signal (top panel) and the Pab1p signal (lower panel). FT, flow-through fraction; E, elution fractions.

Techniques Used: Filtration, Chromatography, Western Blot, Expressing, Purification, Derivative Assay, Flow Cytometry

Cytoplasmic localization and membrane association of Bfr1p. ( A ) Anti-GFP immunofluorescence microscopy of fixed cells expressing either GFP-Bfr1 or Scp160-GFP. Bfr1p (top panel) shows an almost identical distribution to Scp160p (bottom panel), appearing cytoplasmic with an enrichment around the nuclear envelope/ER. Panels to the right demonstrate DAPI staining of nuclear and mitochondrial DNA in the same cells. Arrows indicate clear examples of perinuclear staining. ( B ) Western blot analyses following subcellular fractionation of lysates from yeast expressing HA-Bfr1p and FLAG-Scp160p. Pellets were washed in the presence (right panels) or absence (left panels) of 50 U/ml RNase I. The open arrow indicates the location of an endogenous α-FLAG cross-reacting protein which does not associate with membrane pellets in this assay.
Figure Legend Snippet: Cytoplasmic localization and membrane association of Bfr1p. ( A ) Anti-GFP immunofluorescence microscopy of fixed cells expressing either GFP-Bfr1 or Scp160-GFP. Bfr1p (top panel) shows an almost identical distribution to Scp160p (bottom panel), appearing cytoplasmic with an enrichment around the nuclear envelope/ER. Panels to the right demonstrate DAPI staining of nuclear and mitochondrial DNA in the same cells. Arrows indicate clear examples of perinuclear staining. ( B ) Western blot analyses following subcellular fractionation of lysates from yeast expressing HA-Bfr1p and FLAG-Scp160p. Pellets were washed in the presence (right panels) or absence (left panels) of 50 U/ml RNase I. The open arrow indicates the location of an endogenous α-FLAG cross-reacting protein which does not associate with membrane pellets in this assay.

Techniques Used: Immunofluorescence, Microscopy, Expressing, Staining, Western Blot, Fractionation

RNA-dependent association of Bfr1p with Scp160p-containing mRNP complexes. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast co-expressing FLAG-Scp160p and HA-Bfr1p. ( B ) α-FLAG purification and western blot analyses of S300 void fractions derived from the same strain as in (A), with and without RNase pre-treatment. An α-Pab1p western blot of the same samples is shown as a control. FT, flow-through fraction; E, elution fractions.
Figure Legend Snippet: RNA-dependent association of Bfr1p with Scp160p-containing mRNP complexes. ( A ) S300 gel filtration chromatography followed by western blot analysis of EDTA-treated lysates from yeast co-expressing FLAG-Scp160p and HA-Bfr1p. ( B ) α-FLAG purification and western blot analyses of S300 void fractions derived from the same strain as in (A), with and without RNase pre-treatment. An α-Pab1p western blot of the same samples is shown as a control. FT, flow-through fraction; E, elution fractions.

Techniques Used: Filtration, Chromatography, Western Blot, Expressing, Purification, Derivative Assay, Flow Cytometry

23) Product Images from "In Vivo Expression of Major Histocompatibility Complex Molecules on Oligodendrocytes and Neurons during Viral Infection"

Article Title: In Vivo Expression of Major Histocompatibility Complex Molecules on Oligodendrocytes and Neurons during Viral Infection

Journal: The American Journal of Pathology

doi:

MHC class I expression in mouse spinal cords following MHV V5A13.1 infection. Double immunofluorescence imaged with confocal microscopy was used to identify cells expressing MHC class I (shown in red) and cell type specific markers (green). Colocalization of MHC class I and cell type markers is shown as yellow in the merged panels in the third column. Within or near the edge of a demyelinated lesion at 10–14 dpi, oligodendrocytes ( A1 ) and myelinated processes ( B1 ) labeled with CNP express MHC class I ( A2, B2 ), indicated by yellow structures in A3 and B3 . In B3 , the arrow indicates a yellow process colocalizing CNP and MHC class I. The arrowhead indicates a green process labeled with CNP only. Spinal cord neurons labeled with NSE ( C1, D1 ) also expressed MHC class I ( C2, D2 ), indicated by the yellow cell in C3 and two yellow cells in D3 . Astrocytes labeled with GFAP ( E1 ) did not express MHC class I ( E2 ; merge in E3 ). Microglia expressed MHC class I as indicated by yellow colocalization ( arrow in F3 ) of Mac-1 ( F1 ) and MHC class I ( F2 ). The arrowhead in F3 indicates a blood vessel labeled with MHC class I. MHC class I expression by endothelial cells was confirmed by colocalization of CD31 ( G1 ) and MHC class I ( G2 ) shown with arrow in G3 . Perivascular cells were often seen expressing MHC class I ( arrowhead in G3 ). Rows A and F are three-dimensional reconstructions from 3 to 4 planes 0.2 μm apart. Scale bars, 10 μm.
Figure Legend Snippet: MHC class I expression in mouse spinal cords following MHV V5A13.1 infection. Double immunofluorescence imaged with confocal microscopy was used to identify cells expressing MHC class I (shown in red) and cell type specific markers (green). Colocalization of MHC class I and cell type markers is shown as yellow in the merged panels in the third column. Within or near the edge of a demyelinated lesion at 10–14 dpi, oligodendrocytes ( A1 ) and myelinated processes ( B1 ) labeled with CNP express MHC class I ( A2, B2 ), indicated by yellow structures in A3 and B3 . In B3 , the arrow indicates a yellow process colocalizing CNP and MHC class I. The arrowhead indicates a green process labeled with CNP only. Spinal cord neurons labeled with NSE ( C1, D1 ) also expressed MHC class I ( C2, D2 ), indicated by the yellow cell in C3 and two yellow cells in D3 . Astrocytes labeled with GFAP ( E1 ) did not express MHC class I ( E2 ; merge in E3 ). Microglia expressed MHC class I as indicated by yellow colocalization ( arrow in F3 ) of Mac-1 ( F1 ) and MHC class I ( F2 ). The arrowhead in F3 indicates a blood vessel labeled with MHC class I. MHC class I expression by endothelial cells was confirmed by colocalization of CD31 ( G1 ) and MHC class I ( G2 ) shown with arrow in G3 . Perivascular cells were often seen expressing MHC class I ( arrowhead in G3 ). Rows A and F are three-dimensional reconstructions from 3 to 4 planes 0.2 μm apart. Scale bars, 10 μm.

Techniques Used: Expressing, Infection, Immunofluorescence, Confocal Microscopy, Labeling

MHV V5A13.1 infection induces an inflammatory response and demyelination in mouse spinal cord. Mouse spinal cords taken 10 to 14 days after intracranial infection with MHV have inflammation characterized by expression of MHC class I ( A1 ), MHC class II ( A2 ), and infiltration of CD8+ T cells ( A3 ) compared to PBS-injected mouse spinal cords ( B1–B3 ). In addition, MHV V5A13.1 induces demyelination in ventral-lateral white matter as early as 10 dpi indicated by reduced CNP stain and vacuolization seen in A4 , compared to normal white matter seen in PBS injected mice ( B4 ). Viral antigen can be detected at 10 dpi in spinal cord white matter ( C1 ) and gray matter ( C2 ). Original magnifications: A1–A3 , B1–B3 , 125×; A4 , B4 , 500×; scale bar in C1 , C2 , 50 μm.
Figure Legend Snippet: MHV V5A13.1 infection induces an inflammatory response and demyelination in mouse spinal cord. Mouse spinal cords taken 10 to 14 days after intracranial infection with MHV have inflammation characterized by expression of MHC class I ( A1 ), MHC class II ( A2 ), and infiltration of CD8+ T cells ( A3 ) compared to PBS-injected mouse spinal cords ( B1–B3 ). In addition, MHV V5A13.1 induces demyelination in ventral-lateral white matter as early as 10 dpi indicated by reduced CNP stain and vacuolization seen in A4 , compared to normal white matter seen in PBS injected mice ( B4 ). Viral antigen can be detected at 10 dpi in spinal cord white matter ( C1 ) and gray matter ( C2 ). Original magnifications: A1–A3 , B1–B3 , 125×; A4 , B4 , 500×; scale bar in C1 , C2 , 50 μm.

Techniques Used: Infection, Expressing, Injection, Staining, Mouse Assay

Related Articles

Centrifugation:

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection
Article Snippet: .. After an end-to-end rotation overnight at 4°C, pansorbin cells were washed three times with 1 ml of extraction buffer (100,000 × g for 2 min at 4°C), resuspended in 40 μl of SDS sample buffer , and boiled for 4 min. Pansorbin cells were removed by centrifugation, and protein samples were subjected to SDS–PAGE using 7% polyacrylamide slab gels and further analyzed by Western blot using monoclonal TN-R Abs or pTN-R, secondary HRP-conjugated Abs (Boehringer Mannheim, Mannheim, Germany), and the ECL method for detection (Pierce, Rockford, IL). .. After an end-to-end rotation overnight at 4°C, pansorbin cells were washed three times with 1 ml of extraction buffer (100,000 × g for 2 min at 4°C), resuspended in 40 μl of SDS sample buffer , and boiled for 4 min. Pansorbin cells were removed by centrifugation, and protein samples were subjected to SDS–PAGE using 7% polyacrylamide slab gels and further analyzed by Western blot using monoclonal TN-R Abs or pTN-R, secondary HRP-conjugated Abs (Boehringer Mannheim, Mannheim, Germany), and the ECL method for detection (Pierce, Rockford, IL).

Blocking Assay:

Article Title: Persistent Neuroinflammatory Effects of Serial Exposure to Stress and Methamphetamine on the Blood-Brain Barrier
Article Snippet: .. Membranes were then incubated with primary antibodies (mouse antioccludin, 1:500, (Invitrogen, Carlsbad, CA, Cat. 331500); mouse anti-claudin-5, 1:500, (Invitrogen, Carlsbad, CA, Cat. 352500); mouse anti-β-dystroglycan, 1:500, (Leica Microsystems, Novocastra Reagents, Buffalo Grove, IL, Cat. B-DG-CE); mouse anti-GFAP, 1:2000, (Boehringer Mannheim, Cat. 814369); goat anti-COX-2, 1:500, (Santa Cruz, Cat. sc-1746) mouse anti-α-tubulin, 1:3000, (Sigma, St. Louis, MO, Cat. T6074)) in blocking buffer for approximately 18 hrs at 4 °C. .. Following 3, 5 min, washes with TBS containing Tween-20 (TBS-T) (0.5 % Tween for occuldin, claudin-5, GFAP, COX-2 and α-tubulin; 0.1 % Tween for β-dystroglycan), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse IgG, 1:2500, (Santa Cruz) or rabbit antigoat IgG, 1:2500, (Santa Cruz)) in blocking buffer for 1 hr at room temperature.

Mouse Assay:

Article Title: Genetic dissection of lupus pathogenesis: a recipe for nephrophilic autoantibodies
Article Snippet: .. This increase in total serum IgG was paralleled by significant increases in total serum IgG2a ( P < 0.01), IgG2b ( P < 0.002), and IgG3 ( P < 0.0001) compared with B6 and B6.NZMc1 mice, with the B6.NZMc7 mice exhibiting intermediate levels of these IgG subclasses. ..

Incubation:

Article Title: Persistent Neuroinflammatory Effects of Serial Exposure to Stress and Methamphetamine on the Blood-Brain Barrier
Article Snippet: .. Membranes were then incubated with primary antibodies (mouse antioccludin, 1:500, (Invitrogen, Carlsbad, CA, Cat. 331500); mouse anti-claudin-5, 1:500, (Invitrogen, Carlsbad, CA, Cat. 352500); mouse anti-β-dystroglycan, 1:500, (Leica Microsystems, Novocastra Reagents, Buffalo Grove, IL, Cat. B-DG-CE); mouse anti-GFAP, 1:2000, (Boehringer Mannheim, Cat. 814369); goat anti-COX-2, 1:500, (Santa Cruz, Cat. sc-1746) mouse anti-α-tubulin, 1:3000, (Sigma, St. Louis, MO, Cat. T6074)) in blocking buffer for approximately 18 hrs at 4 °C. .. Following 3, 5 min, washes with TBS containing Tween-20 (TBS-T) (0.5 % Tween for occuldin, claudin-5, GFAP, COX-2 and α-tubulin; 0.1 % Tween for β-dystroglycan), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse IgG, 1:2500, (Santa Cruz) or rabbit antigoat IgG, 1:2500, (Santa Cruz)) in blocking buffer for 1 hr at room temperature.

Article Title: Combined actions of Na+/K+-ATPase, NCX1 and glutamate dependent NMDA receptors in ischemic rat brain penumbra
Article Snippet: .. Membranes were blocked with 5% milk in phosphate buffer solution-T (80 mM Na2 HPO4 , 20 mM NaH2 PO4 , 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4℃ with rabbit anti-Na+ /K+ -ATPase affinity purified polyclonal antibody (Chemicon, Temecula CA, USA, 1: 1,000), rabbit anti-NCX1 affinity purified polyclonal antibody (Chemicon, Temecula CA, USA, 1 : 2,000), rabbit anti-NMDA receptor subunits (NR2A, NR2B) polyclonal antibody (gifted from Moon, 1 : 5,000), mouse anti-neuronal nuclei (NeuN) monoclonal antibody (Chemicon, Temecula CA, USA, 1 : 1,000), mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (Boehringer Mannheim Biochemica, Philadelphia, USA, 1 : 2,000), and mouse anti-2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) monoclonal antibody (Chemicon, Temecula CA, USA, 1: 1,000). .. The sites of antibody-antigen reaction were visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P447 or P448, diluted 1 : 3.000; DAKO, Glostrup, Denmark), an enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) system and exposure to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech, Little Chalfont, UK).

Western Blot:

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection
Article Snippet: .. After an end-to-end rotation overnight at 4°C, pansorbin cells were washed three times with 1 ml of extraction buffer (100,000 × g for 2 min at 4°C), resuspended in 40 μl of SDS sample buffer , and boiled for 4 min. Pansorbin cells were removed by centrifugation, and protein samples were subjected to SDS–PAGE using 7% polyacrylamide slab gels and further analyzed by Western blot using monoclonal TN-R Abs or pTN-R, secondary HRP-conjugated Abs (Boehringer Mannheim, Mannheim, Germany), and the ECL method for detection (Pierce, Rockford, IL). .. After an end-to-end rotation overnight at 4°C, pansorbin cells were washed three times with 1 ml of extraction buffer (100,000 × g for 2 min at 4°C), resuspended in 40 μl of SDS sample buffer , and boiled for 4 min. Pansorbin cells were removed by centrifugation, and protein samples were subjected to SDS–PAGE using 7% polyacrylamide slab gels and further analyzed by Western blot using monoclonal TN-R Abs or pTN-R, secondary HRP-conjugated Abs (Boehringer Mannheim, Mannheim, Germany), and the ECL method for detection (Pierce, Rockford, IL).

Affinity Purification:

Article Title: Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions
Article Snippet: .. Antibodies were diluted as follows: AS 472 1:2000; AS Bianca 1:10,000; AS Florina: 1:20,000; affinity-purified AS 922 1.5 μg/ml; affinity-purified AS 294 0.3 μg/ml; affinity-purified AS α–NgR 0.11 μg/ml; α-BiP 2 μg/ml (Stressgen); mAb α-β-tubulin (Boehringer Mannheim, Mannheim, Germany) 1:200; mAb 9E10 α-myc (Invitrogen) 1:5000; monoclonal hybridoma culture supernatants 1:150. .. Secondary antibodies were HRP-conjugated goat anti-rabbit (Pierce; 1:20,000) and anti-mouse (1:50,000).

Article Title: Combined actions of Na+/K+-ATPase, NCX1 and glutamate dependent NMDA receptors in ischemic rat brain penumbra
Article Snippet: .. Membranes were blocked with 5% milk in phosphate buffer solution-T (80 mM Na2 HPO4 , 20 mM NaH2 PO4 , 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4℃ with rabbit anti-Na+ /K+ -ATPase affinity purified polyclonal antibody (Chemicon, Temecula CA, USA, 1: 1,000), rabbit anti-NCX1 affinity purified polyclonal antibody (Chemicon, Temecula CA, USA, 1 : 2,000), rabbit anti-NMDA receptor subunits (NR2A, NR2B) polyclonal antibody (gifted from Moon, 1 : 5,000), mouse anti-neuronal nuclei (NeuN) monoclonal antibody (Chemicon, Temecula CA, USA, 1 : 1,000), mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (Boehringer Mannheim Biochemica, Philadelphia, USA, 1 : 2,000), and mouse anti-2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) monoclonal antibody (Chemicon, Temecula CA, USA, 1: 1,000). .. The sites of antibody-antigen reaction were visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P447 or P448, diluted 1 : 3.000; DAKO, Glostrup, Denmark), an enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) system and exposure to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech, Little Chalfont, UK).

SDS Page:

Article Title: Tenascin-R Is Antiadhesive for Activated Microglia that Induce Downregulation of the Protein after Peripheral Nerve Injury: a New Role in Neuronal Protection
Article Snippet: .. After an end-to-end rotation overnight at 4°C, pansorbin cells were washed three times with 1 ml of extraction buffer (100,000 × g for 2 min at 4°C), resuspended in 40 μl of SDS sample buffer , and boiled for 4 min. Pansorbin cells were removed by centrifugation, and protein samples were subjected to SDS–PAGE using 7% polyacrylamide slab gels and further analyzed by Western blot using monoclonal TN-R Abs or pTN-R, secondary HRP-conjugated Abs (Boehringer Mannheim, Mannheim, Germany), and the ECL method for detection (Pierce, Rockford, IL). .. After an end-to-end rotation overnight at 4°C, pansorbin cells were washed three times with 1 ml of extraction buffer (100,000 × g for 2 min at 4°C), resuspended in 40 μl of SDS sample buffer , and boiled for 4 min. Pansorbin cells were removed by centrifugation, and protein samples were subjected to SDS–PAGE using 7% polyacrylamide slab gels and further analyzed by Western blot using monoclonal TN-R Abs or pTN-R, secondary HRP-conjugated Abs (Boehringer Mannheim, Mannheim, Germany), and the ECL method for detection (Pierce, Rockford, IL).

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