Journal:

Article Title: CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

doi: 10.1046/j.1365-2567.2001.01325.x

Figure Lengend Snippet: Influence of CR2 and CR1 blockade on C3-fragment deposition and MAC formation on B cells. The percentage of C3d-specific (open columns) and C9-specific (solid columns) fluorescence, respectively, relative to incubation in normal human serum (NHS)/Mg/EGTA, in the absence of blocking antibodies, is given. B cells were incubated under the given conditions in the presence of the CR2-blocking mAb FE8, the CR1-blocking mAb 3D9 or the control mAbs HB135, for CR2 and HB8592, for CR1. The error bars display the 95% confidence intervals for the values obtained with sera from six donors.

Article Snippet: HB135 (IgG2a, anti-CR2, non-function-blocking, 1 μg/ml) and HB8592 (IgG1, anti-CR1, non-function-blocking, 1 μg/ml) were purchased from the American Type Culture Collection (Manassas, VA) and served as controls.

Techniques: Fluorescence, Incubation, Blocking Assay, Control

Journal: NPJ Parkinson's Disease

Article Title: Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

doi: 10.1038/s41531-023-00491-3

Figure Lengend Snippet: a Coronal mouse brain sections stained for PSER129 using TSA. b Representative images of select brain regions with PSER129 immunoreactivity at high magnification. Tissues digested with PK, incubated with anti-PSER129 antibody preabsorbed against PSER129 (PA), processed without primary antibody (-Primary), and from mice lacking alpha-synuclein (KO). Representative images of select brain regions are shown. Scale bars = 50 microns. WT mice, n = 14, KO mice, n = 7. c Enlarged images from select brain regions showing PSER129 staining patterns in each region. d Enlarged image from Fig. b, annotated to show the distribution of PSER129 across layers of the OB. e Western blots of proteins extracted from the OB of WT and KO mice probed for PSER129. Blots were conducted in duplicate, with one blot being pretreated with calf intestine alkaline phosphatase (CIAP). f Blots reprobed for total alpha-synuclein (antibody “SYN1”). “MSA” denotes a positive control sample. For “MSA”, 20 µg of protein extracted from a single striatal section of an MSA brain was separated and blotted. The red arrow denotes a single 15 kDa PSER129 positive band in WT OB. WT, n = 3; KO, n = 3.

Article Snippet: Sections were then incubated with either PSER129 antibody (Abcam, “EP1536Y”) diluted 1:50,000, TBX21 antibody (Santa Cruz Biotechnology) diluted 1:10,000, c-fos antibody (Abcam) diluted 1:10,000, Ywhag antibody (Abcam) diluted 1:5,000, alpha-synuclein antibody (Abcam, “EPR20535”) diluted 1:10,000, ubiquitin antibody (Abcam) 1:10,000 diluted in blocking buffer overnight at 4 °C.

Techniques: Staining, Incubation, Western Blot, Positive Control

Journal: NPJ Parkinson's Disease

Article Title: Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

doi: 10.1038/s41531-023-00491-3

Figure Lengend Snippet: a Summary of approach for measuring PK resistance in situ. Step 1, the location of PSER129 is labeled using TSA. CF-568 dye is covalently bound to tissue (Position “B”). Step 2, low pH and heat are used to remove the antibody complex from the tissue while leaving the CF-568 dye intact. Step 3, tissues are briefly treated with PK to destroy enzyme-accessible epitomes of alpha-synuclein. Step 4, an antibody against total alpha-synuclein is used for TSA labeling of the remaining alpha-synuclein in the sample. (Position “A”). Results allow simultaneous visualization of PK-sensitive and PK-resistant epitopes. b PD brain sections processed with this dual-labeling protocol. High-magnification confocal images of the substantia nigra. The top panels depict results without PK and below with PK digestion (“PK”). c Low magnification confocal images of OB dual-labeled for PSER129 and alpha-synuclein. The top panels show an OB section processed in the absence of PK. The bottom panels depict OB sections processed with PK. d High-magnification images of the mitral cell layer. Expanded high-magnification images depicting a mitral cell body ( e ) and apical dendrite ( f ) in the EPL with (bottom panel) and without PK (top panel). g Distribution of PSER129 and PK-resistant alpha-synuclein in a single OB mitral cell. MCL mitral cell layer, EPL external plexiform layer. (WT mice n = 3, PD substantia nigra = 3).

Article Snippet: Sections were then incubated with either PSER129 antibody (Abcam, “EP1536Y”) diluted 1:50,000, TBX21 antibody (Santa Cruz Biotechnology) diluted 1:10,000, c-fos antibody (Abcam) diluted 1:10,000, Ywhag antibody (Abcam) diluted 1:5,000, alpha-synuclein antibody (Abcam, “EPR20535”) diluted 1:10,000, ubiquitin antibody (Abcam) 1:10,000 diluted in blocking buffer overnight at 4 °C.

Techniques: In Situ, Labeling

Journal: NPJ Parkinson's Disease

Article Title: Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

doi: 10.1038/s41531-023-00491-3

Figure Lengend Snippet: BAR-PSER129 was conducted on brain sections from WT and KO mice. A primary antibody omission control was conducted for each sample (“Neg”), where the samples were processed in the absence of the PSER129 antibody. a Spot blot of the captured proteins were probed for biotinylated proteins or alpha-synuclein. The resulting chemiluminescent detection is shown. b Samples were analyzed by LC-MS/MS. Venn diagram depicts the overlap of identified proteins between samples. 125 proteins were identified exclusively in the BAR-PSER129 WT sample. c Alpha-synuclein identification in the BAR-PSER129 capture sample. Three high-confidence peptides (highlighted yellow) were identified. Spectrum from the most abundant alpha-synuclein peptide identified. (Denoted by an asterisk). d STRING analysis and MCL clustering of the 125 proteins identified. High-confidence functional interactions are shown (>0.7). Proteins lacking high-confidence functional interactions are not shown. Enrichment analysis was performed for each distinct STRING cluster and a consensus-enriched term(s) were manually annotated above each cluster. Protein nodes are colored for easy visualization of each cluster. Open-circle nodes indicate alpha-synuclein interactors previously identified in rat primary cortical neurons using in vivo proximity labeling methods .

Article Snippet: Sections were then incubated with either PSER129 antibody (Abcam, “EP1536Y”) diluted 1:50,000, TBX21 antibody (Santa Cruz Biotechnology) diluted 1:10,000, c-fos antibody (Abcam) diluted 1:10,000, Ywhag antibody (Abcam) diluted 1:5,000, alpha-synuclein antibody (Abcam, “EPR20535”) diluted 1:10,000, ubiquitin antibody (Abcam) 1:10,000 diluted in blocking buffer overnight at 4 °C.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Functional Assay, In Vivo, Labeling

Journal: The American Journal of Pathology

Article Title: Inhibiting the Secreted RGDKGE Collagen Peptide Selectively Controls CD8 + T-Cell Migration on Denatured Collagen-IV and Enhances Their Accumulation in Tumors

doi: 10.1016/j.ajpath.2025.09.008

Figure Lengend Snippet: CD8 + T cells control tumor growth following targeting the RGDKGE collagen peptide. C57BL/6 mice were treated intraperitoneally with function-blocking anti–CD8α-depleting antibody (BP0061) or non-specific control antibodies. A: Example of flow cytometry analysis for CD8 + T cells from spleens of mice treated with non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). B: Quantification of CD8 + T cells from spleens of mice treated with non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). Data represent CD8 + T cells from four mice per group. C: Example of flow cytometry analysis for CD4 + T cells from spleens of mice treated with non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). D: Quantification of CD4 + T cells from spleens of mice treated with either non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). Data represent CD4 + T cells from four mice per group. E: Examples of CD8 + T cells (red) in B16F10 tumor section from either non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8) treated mice. F: Quantification of the mean CD8 + T-cell count from four different tumors from each treatment group using five to seven ×200 microscopic fields from each tumor. Data represent CD8 + T-cell counts from four different tumors from each treatment group. G: Quantification of B16F10 tumor size from control antibody-depleted (Ab Cont) or CD8-depleted (Anti-CD8) mice over 14 days. Data points represent tumor volume from four mice per condition. H: Quantification of B16F10 tumor size from control antibody-depleted mice treated with non-specific control antibody (Ab Cont) or anti-RGDKGE antibody [monoclonal antibody (Mab) XL313] over 14 days. Data points represent tumor volume from eight mice per condition. I: Quantification of B16F10 tumor size from CD8-depleted mice treated with non-specific control antibody (Ab Cont) or anti-RGDKGE antibody (Mab XL313) over 14 days. Data points represent tumor volume from seven to eight mice per condition. Data are given as means ± SEM ( B , D , and F – I ). ∗ P < 0.05, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. Scale bars = 50 μm ( E ). SSC, side scatter.

Article Snippet: Mab XL313, functional blocking anti-CD8α (BP0061) used for depletion, and non-specific control antibodies were acquired from Bio X-Cell (Lebanon, NH).

Techniques: Control, Blocking Assay, Flow Cytometry, Cell Characterization

Journal:

Article Title: Endothelial CD47 interaction with SIRPγ is required for human T-cell transendothelial migration under shear flow conditions in vitro

doi: 10.1182/blood-2008-01-134429

Figure Lengend Snippet: SIRPγ and SIRPα expression in T cells. (A) FACS analysis of resting human CD3+ T cells and PMNs stained with anti-SIRPα, anti-SIRPγ, or anti-MHC class I mAbs (solid histograms). The black solid lines represent a nonbinding isotype control mAb. (B) Equal amounts of total protein from lysates of resting PMNs and T cells were separated by SDS-PAGE under reducing conditions and analyzed by Western blotting for SIRPα expression (top). The membrane was stripped and reprobed with a SIRPγ mAb. The positions of SIRPα and SIRPγ proteins are marked. In addition, PMNs or T-cell lysates were loaded under reducing (lanes 1 and 3 from left) or nonreducing conditions (lanes 2 and 4 from left), electrophoresed, transferred to nitrocellulose membranes, and probed with a rabbit polyclonal Ab, which specifically recognizes the cytoplasmic tail of SIRPα (bottom). (C) FACS analysis of resting (black solid line) and 4 hours of TNF-α (black dotted line) treated HUVECs stained with anti-SIRPα (SE7C2; left), anti-SIRPγ (LSB2.20; right), or an isotype IgG control mAb (gray solid lines). These data are representative of 3 different preparations of cells.

Article Snippet: The following mAbs have been reported previously and were used as purified IgG: C5D5 18 and B6H12 (ATCC, Manassas, VA) are function blocking mAbs to human CD47; a nonblocking mAb against CD47, 2D3 was obtained from Dr Eric Brown (Genentech, South San Francisco, CA); blocking anti-SIRPα (SE7C2) 19 and anti-SIRPγ (LSB2.20) 15 mAbs were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal antibody to the cytoplasmic tail of SIRPα (catalog no. 1125) was obtained from ProSci (Poway, CA); Hec-1 mAb 20 recognizes human VE-cadherin and was obtained from Dr William Muller (Northwestern University, Evanston, IL); anti-MHC class I (W6/32) mAb was from ATCC 21 ; and anti–ICAM-1 function blocking (HU5/3) mAb.

Techniques: Expressing, Staining, Control, SDS Page, Western Blot, Membrane

Journal:

Article Title: Endothelial CD47 interaction with SIRPγ is required for human T-cell transendothelial migration under shear flow conditions in vitro

doi: 10.1182/blood-2008-01-134429

Figure Lengend Snippet: SIRPγ mediates TEM of HUVECs under flow. (A) HUVEC monolayers were prepared as described in Figure 2. Isolated CD3+ T cells were preincubated with SIRPα (SE7C2), SIRPγ (LSB2.20), or a nonblocking mAb to MHC class I and then cells were drawn across TNF-α–stimulated endothelium. The total number of accumulated T cells was determined 10 minutes after perfusion as described in “Methods.” Data are means (± SEM) of 4 separate experiments. (B) T cells were treated with listed mAb (30 μg/mL; SIRPα, SE7C2; SIRPγ, LSB2.20; nonblocking control mAb MHC class I, W6/32) and then were drawn across TNF-α–stimulated endothelium for 10 minutes. The percentage of T cells that transmigrated the endothelium after 10 minutes was determined as detailed in “Methods.” Data are means ± SEM, n = 4 separate experiments; *P = .001. (C) DIC images of live cells in the TEM assay after 10 minutes were prepared as described in “T-cell locomotion and transmigration under flow.” The polarity of T cells on control (W6/32), SIRPγ (LSB2.20), and SIPRα (SEC7C2) mAbs are shown. show normal, migrated CD3+ cells in W6/32-treated monolayers. In contrast, T cells (▶) on SIRPγ-treated T cells exhibit a rounded-up morphology and defects in migratory behavior and show reduced transmigration. (D) The apical migration velocities of T cells on HUVEC monolayers treated with SIRPα (SE7C2), SIRPγ (LSB2.20), or nonblocking control mAb MHC class I mAbs was determined by the Image J software. *P = .01. (E) TNF-α–stimulated HUVEC monolayers were preincubated for 30 minutes with anti-CD47 (B6H12) or SIRPα (SE7C2) mAbs. Medium alone (no additions) or MHC-class I (W6/32) mAb served as controls. The percentage of T cells that transmigrated after 10 minutes was determined as described in “Methods.” Data are means (± SEM) of 4 separate experiments; *P = .001.

Article Snippet: The following mAbs have been reported previously and were used as purified IgG: C5D5 18 and B6H12 (ATCC, Manassas, VA) are function blocking mAbs to human CD47; a nonblocking mAb against CD47, 2D3 was obtained from Dr Eric Brown (Genentech, South San Francisco, CA); blocking anti-SIRPα (SE7C2) 19 and anti-SIRPγ (LSB2.20) 15 mAbs were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal antibody to the cytoplasmic tail of SIRPα (catalog no. 1125) was obtained from ProSci (Poway, CA); Hec-1 mAb 20 recognizes human VE-cadherin and was obtained from Dr William Muller (Northwestern University, Evanston, IL); anti-MHC class I (W6/32) mAb was from ATCC 21 ; and anti–ICAM-1 function blocking (HU5/3) mAb.

Techniques: Isolation, Control, Transmigration Assay, Migration, Software