Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: Demographic and Clinicopathologic Characteristics for LBD and FTD

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques:

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: A: Schematic representations of Rac1 and RAc1b mRNAs showing an extra exon (3b) in Rac1b and the locations of promoters used in RT-PCR. B: Agarose gels showing the levels of PCR products for Rac1 (F1/R1), Rac1b (F2/R1), and actin from heart, liver, brain (normal human frontal cortex), placenta, spleen, small intestine, colon, MCF-7 (human breast adenocarcinoma cell line), MDA MB 231 (breast cancer), renal cancer, and prostate cancer.

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: A: Representative immunoblots of recombinant Rac1b and Rac1 (50 kDa) proteins probed with our generated antibody against Rac1b (SG-4088; left) and with a commercial antibody against Rac1 (ARC03; right). Note that Rac1b antiserum (SG-4088) specifically recognized Rac1b protein and did not cross-react with Rac1, whereas the commercial Rac1 antibody cross-reacted with Rac1b protein. B: Immunoblot from NIH3T3 cell cultures transfected with Myc-tagged Rac1b into the NIT tet off expression vector in the absence (-) or presence of different doses of doxycycline (dox) probed with anti-Rac1b and anti-Myc antibodies. The bands from NIH3T3 cells cultured with the lowest concentration (0.2 pg) or absence of dox showed the highest Rac1b-ir band, which was confirmed by reprobing the blots with the antibody against Myc. Rac1b antiserum also recognized endogenous Rac1b expression in NIH3T3 cells that was not shown with the Myc antibody. C and E: Contrast-phase images of HEK293 cells with Myc-tagged Rac1b vector (C) or empty vector (E), showing the overall cell culture appearance. D and F: Immunofluorescence images of C and E panels, showing Rac1b positive cells (red; D) and the absence of immunostaining for Rac1b (F), respectively. Original magnification, ×10.

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques: Western Blot, Recombinant, Generated, Transfection, Expressing, Plasmid Preparation, Cell Culture, Concentration Assay, Immunofluorescence, Immunostaining

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: A: Representative immunoblot of cortical samples from a NCI and a severe AD case probed with our generated antibody against Rac1b (SG-4088). B: Bright-field image showing the lack of Rac1b immunolabeling in NB neurons after pre-adsorption of the anti-Rac1b antibody (SG-4088) with Rac1b protein. C: Bright-field image of double-immunolabeled Rac1b (black)/p75NTR (brown) and single-labeled p75NTR (brown, black arrow) NB neurons in an AD case. Inset shows higher magnification of a Rac1b/p75NTR neuron shown in panel C (open arrow). Scale bars: 30 μm (B and C); 20 μm (inset).

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques: Western Blot, Generated, Immunolabeling, Adsorption, Labeling

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: Schematics showing the distribution of neurons either dual stained for Rac1b+/p75NTR (red-brown) and singly for p75NTR (green) at similar anteromedial (Ch4am) and anterolateral (Ch4al) levels of the NB in subjects with NCI (A), MCI (B), mild AD (C), and severe AD (D). The specific topographic level of the basal forebrain is depicted in the small coronal drawings seen to the right. Note the increase in double-labeled Rac1b+/p75NTR neurons in Ch4am and Ch4al NB portions in mild and severe AD compared with NCI and MCI. Each square represents a single cell. ac, anterior commissure; AMG, amygdala; Ch4al, cholinergic anterolateral NB portion; Ch4am, cholinergic anteromedial NB portion; Ch4id, cholinergic intermediate-dorsal NB portion; Ch4iv, cholinergic intermediate-ventral NB portion; Fx, fornix; GPe, external globus pallidus; GPi, internal globus pallidus; ic, internal capsule; Put, putamen nucleus.

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques: Staining, Labeling

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: A. Bright-field image of double-immunolabeled Rac1b (black)/p75NTR (brown) and single-labeled p75NTR (brown) neurons in the anteromedial portion (Ch4am) of NB from a severe AD case; inset shows double Rac1b/p75NTR and single p75NTR neurons from panel A (arrow). The specific level of Ch4am is depicted in the schematic drawing shown in panel B. C: Low-power image of double-immunolabeled Rac1b (black)/p75NTR (brown) and single-labeled p75NTR (brown) neurons in the anterolateral portion (Ch4al) of the NB from a severe AD case; inset shows a double Rac1b/p75NTR neuron from panel C (arrow). The specific level Ch4al is depicted in the schematic drawing shown in panel D. E: Image showing double-immunolabeled Rac1b (black)/p75NTR (brown) and single-labeled p75NTR (brown) neurons in the dorsal intermediate NB portion (Ch4id) of severe AD. The specific level of the dorsal-intermediate NB subfield is depicted in the schematic drawing in panel F. G: Rac1b-ir cells with an NFT-like prolife in the temporal cortex of a severe AD case; inset shows a high-power image of a cortical Rac1b-ir neuron from panel G (arrow). H–K: High-power images showing subtypes of neuronal cytoplasmic Rac1b immunoreactivity (black) in p75NTR-positive (brown) NB neurons with a granular (H), filamentous (I and J), or compact spherical (K) appearance in severe AD. L: Twisted Rac1b-positive filaments in the NB in severe AD. M and N: AT8-ir and Alz50-positive neurons and processes in the NB after pre-adsorbing the AT8 and Alz50 antibodies with Rac1b protein, respectively, in severe AD, indicating the specificity of the tau antibody staining. O: Bright-field image of double labeling for Rac1b (black) and p75NTR, showing their colocalization in a neuron (arrow) as well as single p75NTR neurons (brown) in NB in a LBD case; inset shows a higher magnification image of a Rac1b+/p75NTR neuron from panel O (arrow). P: Bright-field image showing double-labeled Rac1b and p75NTR (arrow) and single-labeled p75NTR (brown) NB neurons in a FTD case; inset shows a detail of Rac1b+/p75NTR-positive NB neuron shown in panel P (arrow). ac, anterior commissure; AMG, amygdala; Ch4al, cholinergic anterolateral NB portion; Ch4id, cholinergic intermediate-dorsal NB portion; Ch4iv, cholinergic intermediate-ventral NB portion; Fx, fornix; GPe, external globus pallidus; GPi, internal globus pallidus; ic, internal capsule; Put, putamen nucleus. Scale bars: 100 μm (A and C); 40 μm (inset in A); 120 μm (E); 60 μm (G); 10 μm (H–L, insets in O and P); 30 μm (M–O and inset in C); 20 μm (P and inset in G).

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques: Immunolabeling, Labeling, Staining

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: A–C: Immunofluorescence merged images showing dual-labeled Rac1b (red) and Alz50 (green) NB-positive neurons (orange/yellow) in subjects with NCI (A), MCI (B), and severe AD (C). Note the increase in single (open arrows) and double (white arrows) Rac1b and Alz50-positive neurons in a severe AD case. D–F: Laser confocal merged images of Alz50 (green), AT8 (red), and Rac1b (blue), showing double-labeled Rac1b and Alz50 NB neuron (pale green) in a NCI case (D) and triple-labeled Rac1b, Alz50, and AT8 (pink/white, arrows) NB neurons in MCI (E) and severe AD (F) cases. G–I: Laser confocal merged images of ChAT (red), Alz50 (green), and Rac1b (blue), showing few double-labeled Alz50 and ChAT (yellow) process and neurons in NB in NCI (G; white arrow) and MCI (H; white arrow), whereas single Rac1b-ir neurons (blue) and triple stained for Rac1b, Alz50, and AT8 neuron (white) were seen in AD (I). Note the increase in the extent of double-labeled Alz50 and ChAT neuropil threads (yellow) in AD (I) compared with MCI (H). Scale bars: 40 μm (A, C, D, and E); 50 μm (B, G–I); 30 μm (F).

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques: Immunofluorescence, Labeling, Staining

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: Bar graph showing the mean values of the percentage of Rac1b+/p75NTR-ir Ch4 NB neurons in NCI, MCI, and mild and severe AD. Statistical analysis indicated a significant increase in the percentage of double Rac1b+/p75NTR-ir in NB neurons in mild and severe AD compared with the NCI and MCI groups (*P < 0.05).

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques:

Journal: The American Journal of Pathology

Article Title: Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

doi: 10.1016/j.ajpath.2011.10.027

Figure Lengend Snippet: Linear graphs showing the mean values of the relative density of fetal Alzheimer antigen (FALZ) (A), GNB4 (B), LIPA (C), and CAV2 (D) transcripts in NCI and severe AD obtained from single p75NTR and AD double Rac1b+/p75NTR-positive NB neurons with the use of single-cell gene array technology. Significant down-regulation in FALZ mRNA in p75NTR-ir Ch4 neurons was detected between NCI and AD (A). In addition, down-regulation of GNB4 (B), LIPA (C), and CAV2 (D) transcripts in AD Rac1b+/p75NTR neurons compared with NCI p75NTR-positive NB neurons. Down-regulation of CAV2 (D) was also found between p75NTR-ir and Rac1b+/p75NTR-positive neurons in severe AD (*P < 0.05).

Article Snippet: An anti-Rac1b serum (SG-4088) was produced by Sigma-Genosys (Sigma-Aldrich, St. Louis, MO) according to their standard immunization protocol by coupling the peptide VGETYGKDITSRGKDKPIAC from AD frontal cortex to keyhole limpet hemocyanin.

Techniques:

Journal: PLoS ONE

Article Title: Potential Relevance of α 1 -Adrenergic Receptor Autoantibodies in Refractory Hypertension

doi: 10.1371/journal.pone.0003742

Figure Lengend Snippet: (A) α 1 -AAB isolated from the serum of a patient with refractory hypertension elicited a Ca 2+ signal. (B) The rabbit α 1 -AB gave a similar Ca 2+ signal. (C) A human control IgG preparation was unable to affect intracellular Ca 2+ . Cardiomyocytes were electrically stimulated at 1 Hz, and the peak Ca 2+ was monitored.

Article Snippet: Following, vessel rings were incubated with α 1 -AAB (5 µg in 5 ml PBS buffer), rabbit α 1 -AB (50 µg in 5 ml PBS buffer), rabbit IgG (50 µg in 5 ml PBS buffer, Dunn Labortechnik GmBH, Asbach, Germany) and human control IgG endobulin (50 µg in 5 ml PBS buffer).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Potential Relevance of α 1 -Adrenergic Receptor Autoantibodies in Refractory Hypertension

doi: 10.1371/journal.pone.0003742

Figure Lengend Snippet: A brisk KCl response is documented. (A) Representative experiment showing the contractile response to phenylephrine (PE, 10 nM–10 µM) and rabbit α 1 -AB (50 µg in 5 ml PBS buffer). The PE and antibody responses were similar in kind. (B) Contractile response of human α 1 -AAB isolated from two patients (5 µg in 5 ml PBS buffer) and rabbit α 1 -AB (50 µg in 5 ml PBS buffer) are compared to the PE response (300 nM and 10 µM, respectively) is shown. Contractions are expressed as % KCl response from patient 1 and patient 2. The human antibody responses were less than the low and high-dose PE responses. The rabbit α 1 -AB responses approached the PE responses. Two columns on the right are control human and control rabbit IgG showing no response.

Article Snippet: Following, vessel rings were incubated with α 1 -AAB (5 µg in 5 ml PBS buffer), rabbit α 1 -AB (50 µg in 5 ml PBS buffer), rabbit IgG (50 µg in 5 ml PBS buffer, Dunn Labortechnik GmBH, Asbach, Germany) and human control IgG endobulin (50 µg in 5 ml PBS buffer).

Techniques: Isolation

Journal: BMC Infectious Diseases

Article Title: Evaluation of envelope domain III-based single chimeric tetravalent antigen and monovalent antigen mixtures for the detection of anti-dengue antibodies in human sera

doi: 10.1186/1471-2334-11-64

Figure Lengend Snippet: Comparative analysis of DENV-specific antibodies in patient sera in indirect ELISAs using DENV EDIII-based coating antigens . Anti-DENV IgG (panels A-D) and IgM (panels E-H) antibodies were detected using a mixture of four monovalent EDIIIs (panels A & E), EDIII-T (panels B & F), or b-EDIII-T (panels C, D, G & H) as the coating antigen. The antigens were coated on either polystyrene (panels A, B, C, E, F & G) or streptavidin (panels D & H) plates. The S/Co values of individual DENV-positive and -negative sera, clustered together, are indicated by filled and unfilled circles, respectively. The horizontal line in each panel indicates the cut-off corresponding to S/Co ratio of 1.

Article Snippet: Sera were diluted 1:80 in IgM assay diluent (IgG assay diluents lacking goat serum and KCl) and pre-incubated with Serion Rf-Absorbent as recommended by the manufacturer (Virion/Serion GmbH, Würzburg, Germany) to remove IgG antibodies before using in the assay.

Techniques: