Structured Review

SouthernBiotech anti p68 antibody
Flow cytometry and Western blot of hCMEC/D3 cells after treatment with siRNA specific for <t>p68</t> RNA helicase (abbreviated p68) or non-targeting control siRNA (abbreviated ctrl). There is a significant reduction of the p68 protein in whole cell extract compared to the reference housekeeping protein, β-actin, after 24 h or 48h of transfection with siRNA (100 nM). Treatment with specific or control siRNA does not cause a difference in FACS staining for BB7.2 at both time points. While staining with RL6A at 24h also shows no difference, at 48h the signal is significantly decreased. MFI = median fluorescence intensity.
Anti P68 Antibody, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 85/100, based on 1005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "A novel vascular targeting strategy for brain-derived endothelial cells using a TCR mimic antibody"

Article Title: A novel vascular targeting strategy for brain-derived endothelial cells using a TCR mimic antibody

Journal: Journal of cellular physiology

doi: 10.1002/jcp.22256

Flow cytometry and Western blot of hCMEC/D3 cells after treatment with siRNA specific for p68 RNA helicase (abbreviated p68) or non-targeting control siRNA (abbreviated ctrl). There is a significant reduction of the p68 protein in whole cell extract compared to the reference housekeeping protein, β-actin, after 24 h or 48h of transfection with siRNA (100 nM). Treatment with specific or control siRNA does not cause a difference in FACS staining for BB7.2 at both time points. While staining with RL6A at 24h also shows no difference, at 48h the signal is significantly decreased. MFI = median fluorescence intensity.
Figure Legend Snippet: Flow cytometry and Western blot of hCMEC/D3 cells after treatment with siRNA specific for p68 RNA helicase (abbreviated p68) or non-targeting control siRNA (abbreviated ctrl). There is a significant reduction of the p68 protein in whole cell extract compared to the reference housekeeping protein, β-actin, after 24 h or 48h of transfection with siRNA (100 nM). Treatment with specific or control siRNA does not cause a difference in FACS staining for BB7.2 at both time points. While staining with RL6A at 24h also shows no difference, at 48h the signal is significantly decreased. MFI = median fluorescence intensity.

Techniques Used: Flow Cytometry, Cytometry, Western Blot, Transfection, FACS, Staining, Fluorescence

2) Product Images from "RNA Interference in J774 Macrophages Reveals a Role for Coronin 1 in Mycobacterial Trafficking but Not in Actin-dependent Processes"

Article Title: RNA Interference in J774 Macrophages Reveals a Role for Coronin 1 in Mycobacterial Trafficking but Not in Actin-dependent Processes

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E07-07-0640

Localization of coronin 1 and F-actin in macrophages. Macrophages (J774) were seeded on coverslips, and they were left untreated (A–D) or activated with 100 nM PMA (E–L). After fixation in 3% paraformaldehyde and permeabilization with 0.1% saponin/2% BSA in PBS, cells were stained with anti-coronin 1 antiserum followed by Texas Red-conjugated secondary antibodies (B, F, and J) or phalloidin-FITC (C, G, and K). Coronin 1 and F-actin colocalized at sites of pseudopod formation (D), at the leading edge (H), and at membrane ruffles (L). Bar, 10 μm.
Figure Legend Snippet: Localization of coronin 1 and F-actin in macrophages. Macrophages (J774) were seeded on coverslips, and they were left untreated (A–D) or activated with 100 nM PMA (E–L). After fixation in 3% paraformaldehyde and permeabilization with 0.1% saponin/2% BSA in PBS, cells were stained with anti-coronin 1 antiserum followed by Texas Red-conjugated secondary antibodies (B, F, and J) or phalloidin-FITC (C, G, and K). Coronin 1 and F-actin colocalized at sites of pseudopod formation (D), at the leading edge (H), and at membrane ruffles (L). Bar, 10 μm.

Techniques Used: Staining

Analysis of J774 cell lines depleted for coronin 1. (A) Equal amounts of wild-type J774 macrophages or the indicated stably transfected cells were lysed in Laemmli sample buffer, and coronin 1 expression was analyzed by immunoblotting after SDS-PAGE by using anti-coronin 1 and anti-actin antibodies. Clones 3–10 and 10–11 express pEGFP-C1::siRNAm CORO1nt198-216; clone 21 expresses the control construct pEGFP-C1::siRNAhCORO1nt 594-612. (B) Wild-type J774macrophages or the indicated stable clones were homogenized, and the postnuclear supernatant was subjected to ultracentrifugation (100,000 × g for 30 min). Actin distribution between membranes (pellet, p) and cytosol (supernatant, s) was analyzed by SDS-PAGE and subsequent immunoblotting by using anti-actin antibody. Protein equivalents were loaded on SDS-PAGE. (C) Analysis of actin distribution between a Triton X-100–insoluble fraction (p) and cytosol (s) by immunoblotting as described in Materials and Methods .
Figure Legend Snippet: Analysis of J774 cell lines depleted for coronin 1. (A) Equal amounts of wild-type J774 macrophages or the indicated stably transfected cells were lysed in Laemmli sample buffer, and coronin 1 expression was analyzed by immunoblotting after SDS-PAGE by using anti-coronin 1 and anti-actin antibodies. Clones 3–10 and 10–11 express pEGFP-C1::siRNAm CORO1nt198-216; clone 21 expresses the control construct pEGFP-C1::siRNAhCORO1nt 594-612. (B) Wild-type J774macrophages or the indicated stable clones were homogenized, and the postnuclear supernatant was subjected to ultracentrifugation (100,000 × g for 30 min). Actin distribution between membranes (pellet, p) and cytosol (supernatant, s) was analyzed by SDS-PAGE and subsequent immunoblotting by using anti-actin antibody. Protein equivalents were loaded on SDS-PAGE. (C) Analysis of actin distribution between a Triton X-100–insoluble fraction (p) and cytosol (s) by immunoblotting as described in Materials and Methods .

Techniques Used: Stable Transfection, Transfection, Expressing, SDS Page, Clone Assay, Construct

Depletion of coronin 1 by RNA interference. Macrophages (J774) transiently transfected with pEGFP-C1::siRNAmCORO1nt198-216 (A) or pSUPER::siRNAmCORO1nt198-216 (B and C) were seeded on coverslips, fixed, and permeabilized on day 4. Cells were stained for coronin 1 (Texas Red-labeled secondary antibody) and tubulin (Alexa Fluor 633 secondary antibody) (A) or coronin 1 (Texas Red-labeled secondary antibody) and phalloidin-FITC (B). In C, wild-type J774 or the indicated stable clones grown on 10-well Teflon-coated glass slides were activated with 100 nM PMA for 20 min and subsequently stained with phalloidin-FITC to visualize the actin cytoskeleton. Bar, 10 μm.
Figure Legend Snippet: Depletion of coronin 1 by RNA interference. Macrophages (J774) transiently transfected with pEGFP-C1::siRNAmCORO1nt198-216 (A) or pSUPER::siRNAmCORO1nt198-216 (B and C) were seeded on coverslips, fixed, and permeabilized on day 4. Cells were stained for coronin 1 (Texas Red-labeled secondary antibody) and tubulin (Alexa Fluor 633 secondary antibody) (A) or coronin 1 (Texas Red-labeled secondary antibody) and phalloidin-FITC (B). In C, wild-type J774 or the indicated stable clones grown on 10-well Teflon-coated glass slides were activated with 100 nM PMA for 20 min and subsequently stained with phalloidin-FITC to visualize the actin cytoskeleton. Bar, 10 μm.

Techniques Used: Transfection, Staining, Labeling, Clone Assay

Trafficking and survival in wild-type and coronin 1-depleted macrophages. (A) Macrophages were infected with  M. bovis  BCG, chased for 3 h, and then fixed with methanol and stained for lysosome-associated membrane protein (LAMP)1 (568) or mycobacteria (488). Cells were analyzed by confocal microscopy. (B and C) Quantitation of the lysosomal delivery of wild-type or PknG-deficient  M. bovis  BCG in the different macrophage clones indicated as determined by confocal microscopy (n ∼ 150). The error bars represent the SD values from three independent experiments. (D) Survival of mycobacteria in the macrophage clones indicated as determined by incorporation of tritiated uracil. The error bars represent SD values from triplicate values, and the data shown are representative of at least three independent experiments.
Figure Legend Snippet: Trafficking and survival in wild-type and coronin 1-depleted macrophages. (A) Macrophages were infected with M. bovis BCG, chased for 3 h, and then fixed with methanol and stained for lysosome-associated membrane protein (LAMP)1 (568) or mycobacteria (488). Cells were analyzed by confocal microscopy. (B and C) Quantitation of the lysosomal delivery of wild-type or PknG-deficient M. bovis BCG in the different macrophage clones indicated as determined by confocal microscopy (n ∼ 150). The error bars represent the SD values from three independent experiments. (D) Survival of mycobacteria in the macrophage clones indicated as determined by incorporation of tritiated uracil. The error bars represent SD values from triplicate values, and the data shown are representative of at least three independent experiments.

Techniques Used: Infection, Staining, Confocal Microscopy, Quantitation Assay, Clone Assay

Expression of coronin 1-7 in cell lines as determined by RT-PCR. Total RNA was extracted from J774 and RAW264.7 macrophages, reversely transcribed, and analyzed for the presence of coronin 1-7 transcripts by PCR. Expression of coronin 1-7 in J774 (A) and RAW264.7 (B) cells. RT, reverse transcriptase. For immunoblotting, equal cell numbers were loaded per well from the cell lines indicated in C after denaturation in Laemmli sample buffer. Blots were probed for the coronin isoforms indicated using anti-tubulin antibodies as a control.
Figure Legend Snippet: Expression of coronin 1-7 in cell lines as determined by RT-PCR. Total RNA was extracted from J774 and RAW264.7 macrophages, reversely transcribed, and analyzed for the presence of coronin 1-7 transcripts by PCR. Expression of coronin 1-7 in J774 (A) and RAW264.7 (B) cells. RT, reverse transcriptase. For immunoblotting, equal cell numbers were loaded per well from the cell lines indicated in C after denaturation in Laemmli sample buffer. Blots were probed for the coronin isoforms indicated using anti-tubulin antibodies as a control.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

3) Product Images from "Smoothelin, a new marker to determine the origin of liver fibrogenic cells"

Article Title: Smoothelin, a new marker to determine the origin of liver fibrogenic cells

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.v19.i48.9343

α-smooth muscle actin and smoothelin expression in normal, fibrotic and cirrhotic livers. A and B: In the normal liver, α-smooth muscle actin (A) and smoothelin (B) show similar expression in tunica media smooth muscle cells of the portal
Figure Legend Snippet: α-smooth muscle actin and smoothelin expression in normal, fibrotic and cirrhotic livers. A and B: In the normal liver, α-smooth muscle actin (A) and smoothelin (B) show similar expression in tunica media smooth muscle cells of the portal

Techniques Used: Expressing

α-smooth muscle actin and smoothelin expression in a cutaneous hypertrophic scar. A: Following a burn injury, a retractile hypertrophic scar was observed (from Vincent Casoli, Plastic Surgery and Burns Unit, University Hospital of Bordeaux, France);
Figure Legend Snippet: α-smooth muscle actin and smoothelin expression in a cutaneous hypertrophic scar. A: Following a burn injury, a retractile hypertrophic scar was observed (from Vincent Casoli, Plastic Surgery and Burns Unit, University Hospital of Bordeaux, France);

Techniques Used: Expressing

Cellular co-localization of α-smooth muscle actin and smoothelin in a fibrotic liver. A: α-smooth muscle actin expression (green); B: Smoothelin expression (red); C: Merged (nuclei are stained with DAPI). Smoothelin-expressing cells also
Figure Legend Snippet: Cellular co-localization of α-smooth muscle actin and smoothelin in a fibrotic liver. A: α-smooth muscle actin expression (green); B: Smoothelin expression (red); C: Merged (nuclei are stained with DAPI). Smoothelin-expressing cells also

Techniques Used: Expressing, Staining

α-Smooth muscle actin and smoothelin expression in hepatocellular carcinoma. A and B: α-smooth muscle actin (A) and smoothelin (B) are expressed similarly in the stroma reaction surrounding the tumor (arrows) and around tumoral hepatocytes;
Figure Legend Snippet: α-Smooth muscle actin and smoothelin expression in hepatocellular carcinoma. A and B: α-smooth muscle actin (A) and smoothelin (B) are expressed similarly in the stroma reaction surrounding the tumor (arrows) and around tumoral hepatocytes;

Techniques Used: Expressing

4) Product Images from "Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac–/ICAM-1-dependent mechanism"

Article Title: Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac–/ICAM-1-dependent mechanism

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

doi: 10.4049/jimmunol.0802167

Interaction of TNFα-matured DCs with ICAM-1 expressed by LECs decreases expression of CD86 in the absence of antigen
Figure Legend Snippet: Interaction of TNFα-matured DCs with ICAM-1 expressed by LECs decreases expression of CD86 in the absence of antigen

Techniques Used: Expressing

Expression of ICAM-1 on collecting lymphatic vessels and on lymphatic sinuses in mouse lymph node
Figure Legend Snippet: Expression of ICAM-1 on collecting lymphatic vessels and on lymphatic sinuses in mouse lymph node

Techniques Used: Expressing

Expression of ICAM-1 on LECs in vitro
Figure Legend Snippet: Expression of ICAM-1 on LECs in vitro

Techniques Used: Expressing, In Vitro

imDCs bind ICAM-1 via Mac-1
Figure Legend Snippet: imDCs bind ICAM-1 via Mac-1

Techniques Used:

The effect of ICAM-1 deficiency on CD86 expression by DCs migrated into regional lymph nodes
Figure Legend Snippet: The effect of ICAM-1 deficiency on CD86 expression by DCs migrated into regional lymph nodes

Techniques Used: Expressing

5) Product Images from "Testosterone is an endogenous regulator of BAFF and splenic B cell number"

Article Title: Testosterone is an endogenous regulator of BAFF and splenic B cell number

Journal: Nature Communications

doi: 10.1038/s41467-018-04408-0

Testosterone regulates splenic catecholamine levels. a Noradrenaline and b dopamine concentration in spleens of castrated (ORX; n = 6) and sham-operated ( n = 5) male mice. c Quantification of tyrosine hydroxylase (TH)-stained area in control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) male mice, expressed as percentage of follicle area. n = 7/group. d–f Sections of spleens from control and G-ARKO mice. Turquoise, nerve structures (tyrosine hydroxylase; TH); red, fibroblastic reticular cells (FRCs) and vascular structures (smooth muscle a-actin; SMA). B cells (IgD) are stained white in e and BAFF is stained white in f . Scale bar, 100 μm. g Higher-magnification image of SMA-positive FRC (red) and TH-stained nerve structures (turquoise); asterisk indicates area of co-localization. Scale bar, 20 μm. Bars indicate means; circles represent individual mice. * P
Figure Legend Snippet: Testosterone regulates splenic catecholamine levels. a Noradrenaline and b dopamine concentration in spleens of castrated (ORX; n = 6) and sham-operated ( n = 5) male mice. c Quantification of tyrosine hydroxylase (TH)-stained area in control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) male mice, expressed as percentage of follicle area. n = 7/group. d–f Sections of spleens from control and G-ARKO mice. Turquoise, nerve structures (tyrosine hydroxylase; TH); red, fibroblastic reticular cells (FRCs) and vascular structures (smooth muscle a-actin; SMA). B cells (IgD) are stained white in e and BAFF is stained white in f . Scale bar, 100 μm. g Higher-magnification image of SMA-positive FRC (red) and TH-stained nerve structures (turquoise); asterisk indicates area of co-localization. Scale bar, 20 μm. Bars indicate means; circles represent individual mice. * P

Techniques Used: Concentration Assay, Mouse Assay, Staining, Knock-Out

6) Product Images from "Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia"

Article Title: Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia

Journal: Oncotarget

doi: 10.18632/oncotarget.25951

Lyn and c-Cbl ubiquitination ( A ) The lysates obtained from leukemic B cells of CLL patients ( n = 10), before and after 5 minutes of IgM and 3 minutes of IgD stimuli (10ng/mL), were immunoprecipitated with anti-Ubiquitin antibody (IP Ubi). Immunocomplexes (IP) and whole lysate (WL) were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl, anti–Lyn and, after stripping the membrane, with anti-ubiquitin-HRP supplied in ubiquitin-immunoprecipitation kit. ( B ) Western blotting (10% Acrylamide/Bis-acrylamide) of CLL B lymphocytes is representative of all 10 samples analysed. The lysates obtained from leukemic B cells, before and after IgM and IgD stimuli, were analyzed by immunostaining with antibody against pCblY700, c-Cbl, pLynY396, Lyn and β-actin. ( C ) Proximity ligation assay (PLA) was performed on CLL and normal B cells (Cnt). Rabbit polyclonal anti-Lyn and mouse monoclonal anti-ubiquitin were used as primary Abs. Positive PLA signals are visualized as green fluorescent spots, nuclei are in blue (DAPI). Images are representative of three independent experiments, and were acquired with zoom 3 using 60X/1.35NA oil immersion objective. Z-projections of 15 slices are shown. Scale bar: 5 µm.
Figure Legend Snippet: Lyn and c-Cbl ubiquitination ( A ) The lysates obtained from leukemic B cells of CLL patients ( n = 10), before and after 5 minutes of IgM and 3 minutes of IgD stimuli (10ng/mL), were immunoprecipitated with anti-Ubiquitin antibody (IP Ubi). Immunocomplexes (IP) and whole lysate (WL) were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl, anti–Lyn and, after stripping the membrane, with anti-ubiquitin-HRP supplied in ubiquitin-immunoprecipitation kit. ( B ) Western blotting (10% Acrylamide/Bis-acrylamide) of CLL B lymphocytes is representative of all 10 samples analysed. The lysates obtained from leukemic B cells, before and after IgM and IgD stimuli, were analyzed by immunostaining with antibody against pCblY700, c-Cbl, pLynY396, Lyn and β-actin. ( C ) Proximity ligation assay (PLA) was performed on CLL and normal B cells (Cnt). Rabbit polyclonal anti-Lyn and mouse monoclonal anti-ubiquitin were used as primary Abs. Positive PLA signals are visualized as green fluorescent spots, nuclei are in blue (DAPI). Images are representative of three independent experiments, and were acquired with zoom 3 using 60X/1.35NA oil immersion objective. Z-projections of 15 slices are shown. Scale bar: 5 µm.

Techniques Used: Immunoprecipitation, SDS Page, Stripping Membranes, Western Blot, Immunostaining, Proximity Ligation Assay

c-Cbl and Lyn were not associated neither after BCR engagement ( A ) Evaluation of mRNA Lyn expression levels by real-time PCR in 14 normal B lymphocytes (Cnt) and 23 neoplastic B cells (CLL). ( B ) Pearson correlation coefficient between Lyn and c-Cbl protein expression obtained from densitometric analysis of all 40 CLL B lymphocytes from CLL patients. ( C ) The lysates obtained from leukemic B cells of CLL patients ( n = 15), before and after 5 minutes of IgM and 3 minutes of IgD stimuli (10 ng/mL), were immunoprecipitated with anti-Lyn (IP Lyn) and anti-c-Cbl antibody (IP c-Cbl). Immunocomplexes (IP) and whole lysate (WL) were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl and anti–Lyn. ( D ) PLA was performed on CLL and normal B cells (Cnt). Rabbit polyclonal anti-Lyn and mouse monoclonal anti-c-Cbl were used as primary Abs. Positive PLA signals are visualized as green fluorescent spots, nuclei are in blue (DAPI). Images are representative of three independent experiments, and were acquired with zoom 4 using 60X/1.35NA oil immersion objective. Z-projections of 15 slices are shown. Scale bar: 5 µm. ( E ) CLL B cells ( n = 5) were treated for 6 h with 17-DMAG (100 µM) and the cytosol, isolated as in panel D of Figure 1 , was immunoprecipitated with anti-Lyn antibody before and after anti-IgM (10 ng/mL) and anti-IgD (10 ng/mL) stimuli. Immunocomplexes were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl, anti-HSP90 and anti-Lyn.
Figure Legend Snippet: c-Cbl and Lyn were not associated neither after BCR engagement ( A ) Evaluation of mRNA Lyn expression levels by real-time PCR in 14 normal B lymphocytes (Cnt) and 23 neoplastic B cells (CLL). ( B ) Pearson correlation coefficient between Lyn and c-Cbl protein expression obtained from densitometric analysis of all 40 CLL B lymphocytes from CLL patients. ( C ) The lysates obtained from leukemic B cells of CLL patients ( n = 15), before and after 5 minutes of IgM and 3 minutes of IgD stimuli (10 ng/mL), were immunoprecipitated with anti-Lyn (IP Lyn) and anti-c-Cbl antibody (IP c-Cbl). Immunocomplexes (IP) and whole lysate (WL) were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl and anti–Lyn. ( D ) PLA was performed on CLL and normal B cells (Cnt). Rabbit polyclonal anti-Lyn and mouse monoclonal anti-c-Cbl were used as primary Abs. Positive PLA signals are visualized as green fluorescent spots, nuclei are in blue (DAPI). Images are representative of three independent experiments, and were acquired with zoom 4 using 60X/1.35NA oil immersion objective. Z-projections of 15 slices are shown. Scale bar: 5 µm. ( E ) CLL B cells ( n = 5) were treated for 6 h with 17-DMAG (100 µM) and the cytosol, isolated as in panel D of Figure 1 , was immunoprecipitated with anti-Lyn antibody before and after anti-IgM (10 ng/mL) and anti-IgD (10 ng/mL) stimuli. Immunocomplexes were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl, anti-HSP90 and anti-Lyn.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Proximity Ligation Assay, Isolation

Analysis of c-Cbl phosphorylation and its association with PI3K regulatory subunit p85 ( A ) Densitometric ananlysis of c-CblY700/c-CblTot ratio in 24 CLL patients before (Alone) and after 5 minutes of IgM (IgM) and 3 minutes of IgD (IgD). Data obtained were evaluated for their statistical significance with pair Student’s t test ( **** p
Figure Legend Snippet: Analysis of c-Cbl phosphorylation and its association with PI3K regulatory subunit p85 ( A ) Densitometric ananlysis of c-CblY700/c-CblTot ratio in 24 CLL patients before (Alone) and after 5 minutes of IgM (IgM) and 3 minutes of IgD (IgD). Data obtained were evaluated for their statistical significance with pair Student’s t test ( **** p

Techniques Used:

7) Product Images from "Antigen activation and impaired Fas-induced death-inducing signaling complex formation in T-large-granular lymphocyte leukemia"

Article Title: Antigen activation and impaired Fas-induced death-inducing signaling complex formation in T-large-granular lymphocyte leukemia

Journal: Blood

doi: 10.1182/blood-2007-06-093823

DISC formation in PBMC from LGL leukemia patients and from healthy donors . (A) Fas-mediated DISC formation was determined after 45 minutes and after 6 hours of cross-linking with (500 ng/mL) anti-Fas (APO-1, IgG3) antibody (lanes 1, 3, 4, 6-9, 11-14) in samples from 5 patients with LGL leukemia. Cells were cultured for 24 hours with medium in the absence (−, lanes 1, 3, 6, 7, and 11) and presence of 500 IU/mL IL-2 (+, lanes 2, 4, 5, 8-10, 12, 13) for 24 hours, or PHA (1 μg/mL) plus IL-2 (500 IU/mL) for 5 to 7 days (indicated as PHA in lane 14). IgG3 isotype control antibody (500 ng/mL) was added to demonstrate specificity of protein interactions with the FasR (lanes 2, 5, and 10). After immunoprecipitation and gel electrophoresis, Western blot analysis was performed to detect caspase-8 (top panel) and FADD (bottom panel) in these FasR immunoprecipitates. Immunoprecipitation after anti-Fas antibody cross-linking of H9 cells (T-cell leukemia cell line) was used as a positive control. (B) Whole-cell lysates were prepared from a fraction of cells studied in these immunoprecipitation experiments after 1 and 6 hours of cross-linking with anti-Fas (Apo-1) antibody and isotype control (IgG3). Results shown represent the means (± SEM) of caspase-8, and -3/7 activities that were determined by a fluorometric enzyme activity assay. Protein bands for full-length caspase-8 (casp-8), FADD, and an activated cleaved product of caspase-8 (Ac casp-8) are indicated. NS = nonspecific band observed with the anti-caspase-8 antibody.
Figure Legend Snippet: DISC formation in PBMC from LGL leukemia patients and from healthy donors . (A) Fas-mediated DISC formation was determined after 45 minutes and after 6 hours of cross-linking with (500 ng/mL) anti-Fas (APO-1, IgG3) antibody (lanes 1, 3, 4, 6-9, 11-14) in samples from 5 patients with LGL leukemia. Cells were cultured for 24 hours with medium in the absence (−, lanes 1, 3, 6, 7, and 11) and presence of 500 IU/mL IL-2 (+, lanes 2, 4, 5, 8-10, 12, 13) for 24 hours, or PHA (1 μg/mL) plus IL-2 (500 IU/mL) for 5 to 7 days (indicated as PHA in lane 14). IgG3 isotype control antibody (500 ng/mL) was added to demonstrate specificity of protein interactions with the FasR (lanes 2, 5, and 10). After immunoprecipitation and gel electrophoresis, Western blot analysis was performed to detect caspase-8 (top panel) and FADD (bottom panel) in these FasR immunoprecipitates. Immunoprecipitation after anti-Fas antibody cross-linking of H9 cells (T-cell leukemia cell line) was used as a positive control. (B) Whole-cell lysates were prepared from a fraction of cells studied in these immunoprecipitation experiments after 1 and 6 hours of cross-linking with anti-Fas (Apo-1) antibody and isotype control (IgG3). Results shown represent the means (± SEM) of caspase-8, and -3/7 activities that were determined by a fluorometric enzyme activity assay. Protein bands for full-length caspase-8 (casp-8), FADD, and an activated cleaved product of caspase-8 (Ac casp-8) are indicated. NS = nonspecific band observed with the anti-caspase-8 antibody.

Techniques Used: Cell Culture, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Positive Control, Enzyme Activity Assay

8) Product Images from "αv Integrins regulate germinal center B cell responses through noncanonical autophagy"

Article Title: αv Integrins regulate germinal center B cell responses through noncanonical autophagy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI99597

Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, MANN-WHITNEY

9) Product Images from "Expression and function of the TL1A/DR3 axis in chronic lymphocytic leukemia"

Article Title: Expression and function of the TL1A/DR3 axis in chronic lymphocytic leukemia

Journal: Oncotarget

doi:

DR3 surface expression in CLL cells A. Representative example of DR3 flow cytometry analysis. Dashed line: isotypic control; regular line: anti-DR3 signal. B. Time course analysis of DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 8). C. Surface DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 36). Data are expressed as DR3-expression median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI). Comparison between unstimulated and anti-IgM-stimulated CLL cells was performed by the two-sample Wilcoxon signed rank sum test. D. Comparison of DR3 expression between B cells from CLL samples ( n = 36) and age-matched healthy donors ( n = 10). Lines represent median values. Comparison was performed using the Mann-Whitney test. ns = not significant. E. Western blot analysis of cell lysates from purified CLL cells ( n = 4), in unstimulated and anti-IgM stimulated conditions. Level of DR3 induction after anti-IgM stimulation is reported as fold change. F. Representative example of DR3 immunofluorescence analysis in CLL lymph-node tissue sections ( n = 2). Panel A: pseudocolour image of DR3 (200×); Panel B: pseudocolour image of CD23 (200×); Panel C: merged pseudocolour image of CD23 (green), DR3 (red) and DNA (blu) (200×).
Figure Legend Snippet: DR3 surface expression in CLL cells A. Representative example of DR3 flow cytometry analysis. Dashed line: isotypic control; regular line: anti-DR3 signal. B. Time course analysis of DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 8). C. Surface DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 36). Data are expressed as DR3-expression median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI). Comparison between unstimulated and anti-IgM-stimulated CLL cells was performed by the two-sample Wilcoxon signed rank sum test. D. Comparison of DR3 expression between B cells from CLL samples ( n = 36) and age-matched healthy donors ( n = 10). Lines represent median values. Comparison was performed using the Mann-Whitney test. ns = not significant. E. Western blot analysis of cell lysates from purified CLL cells ( n = 4), in unstimulated and anti-IgM stimulated conditions. Level of DR3 induction after anti-IgM stimulation is reported as fold change. F. Representative example of DR3 immunofluorescence analysis in CLL lymph-node tissue sections ( n = 2). Panel A: pseudocolour image of DR3 (200×); Panel B: pseudocolour image of CD23 (200×); Panel C: merged pseudocolour image of CD23 (green), DR3 (red) and DNA (blu) (200×).

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY, Western Blot, Purification, Immunofluorescence

10) Product Images from "Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration"

Article Title: Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089542

MSC phenotypic characterization of human skin-derived cells. (A) Morphological analysis of skin-derived cells by phase contrast microscopy. (B) MTS cell proliferation/viability assay. (C) Osteogenic and (D) adipogenic differentiation. (C) Cells cultured in inductive medium formed Alizarin Red S-stained mineralized nodules and (D) Oil red O-stained lipid clusters. (E) Flow cytometry analysis of hematopoietic (CD34, CD45) and MSC (CD90, CD73, CD105) markers. Specific markers are shown by black curves and controls by gray curves. ***p
Figure Legend Snippet: MSC phenotypic characterization of human skin-derived cells. (A) Morphological analysis of skin-derived cells by phase contrast microscopy. (B) MTS cell proliferation/viability assay. (C) Osteogenic and (D) adipogenic differentiation. (C) Cells cultured in inductive medium formed Alizarin Red S-stained mineralized nodules and (D) Oil red O-stained lipid clusters. (E) Flow cytometry analysis of hematopoietic (CD34, CD45) and MSC (CD90, CD73, CD105) markers. Specific markers are shown by black curves and controls by gray curves. ***p

Techniques Used: Derivative Assay, Microscopy, Viability Assay, Cell Culture, Staining, Flow Cytometry, Cytometry

Immunophenotypic profile of human SD-MSCs cultured in Integra and Pelnac. Flow cytometry analysis of SD-MSCs (CD90, CD73, CD105) and hematopoietic (CD45) markers in SD-MSCs after 3 days of culture in Integra (upper panel) and Pelnac (lower panel). Curves in black show the specific markers, and gray curves correspond to controls.
Figure Legend Snippet: Immunophenotypic profile of human SD-MSCs cultured in Integra and Pelnac. Flow cytometry analysis of SD-MSCs (CD90, CD73, CD105) and hematopoietic (CD45) markers in SD-MSCs after 3 days of culture in Integra (upper panel) and Pelnac (lower panel). Curves in black show the specific markers, and gray curves correspond to controls.

Techniques Used: Cell Culture, Flow Cytometry, Cytometry

Multilineage potential of SD-MSCs. (A) Gene expression profile by RT-PCR of SD-MSCs cultivated in standard medium. (B–E) Immunofluorescence staining of (B) CD105 and (C) Fibronectin. (D–F) Double-staining of α-SMA and βIII-Tubulin, (G–I) α-SMA and Nestin and (J–L) βIII-Tubulin and Nestin co-expression. (F, I and L): Merged pictures of D–E, G–H, J–K, respectively. Cell nuclei were stained with DAPI (blue). Arrows: Nestin nuclear staining. Scale bar: 50 µm.
Figure Legend Snippet: Multilineage potential of SD-MSCs. (A) Gene expression profile by RT-PCR of SD-MSCs cultivated in standard medium. (B–E) Immunofluorescence staining of (B) CD105 and (C) Fibronectin. (D–F) Double-staining of α-SMA and βIII-Tubulin, (G–I) α-SMA and Nestin and (J–L) βIII-Tubulin and Nestin co-expression. (F, I and L): Merged pictures of D–E, G–H, J–K, respectively. Cell nuclei were stained with DAPI (blue). Arrows: Nestin nuclear staining. Scale bar: 50 µm.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Double Staining

11) Product Images from "Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia"

Article Title: Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia

Journal: Oncotarget

doi: 10.18632/oncotarget.25951

Lyn and c-Cbl ubiquitination ( A ) The lysates obtained from leukemic B cells of CLL patients ( n = 10), before and after 5 minutes of IgM and 3 minutes of IgD stimuli (10ng/mL), were immunoprecipitated with anti-Ubiquitin antibody (IP Ubi). Immunocomplexes (IP) and whole lysate (WL) were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl, anti–Lyn and, after stripping the membrane, with anti-ubiquitin-HRP supplied in ubiquitin-immunoprecipitation kit. ( B ) Western blotting (10% Acrylamide/Bis-acrylamide) of CLL B lymphocytes is representative of all 10 samples analysed. The lysates obtained from leukemic B cells, before and after IgM and IgD stimuli, were analyzed by immunostaining with antibody against pCblY700, c-Cbl, pLynY396, Lyn and β-actin. ( C ) Proximity ligation assay (PLA) was performed on CLL and normal B cells (Cnt). Rabbit polyclonal anti-Lyn and mouse monoclonal anti-ubiquitin were used as primary Abs. Positive PLA signals are visualized as green fluorescent spots, nuclei are in blue (DAPI). Images are representative of three independent experiments, and were acquired with zoom 3 using 60X/1.35NA oil immersion objective. Z-projections of 15 slices are shown. Scale bar: 5 µm.
Figure Legend Snippet: Lyn and c-Cbl ubiquitination ( A ) The lysates obtained from leukemic B cells of CLL patients ( n = 10), before and after 5 minutes of IgM and 3 minutes of IgD stimuli (10ng/mL), were immunoprecipitated with anti-Ubiquitin antibody (IP Ubi). Immunocomplexes (IP) and whole lysate (WL) were loaded in SDS-PAGE (10% Acrylamide/Bis-acrylamide) and then probed with anti-c-Cbl, anti–Lyn and, after stripping the membrane, with anti-ubiquitin-HRP supplied in ubiquitin-immunoprecipitation kit. ( B ) Western blotting (10% Acrylamide/Bis-acrylamide) of CLL B lymphocytes is representative of all 10 samples analysed. The lysates obtained from leukemic B cells, before and after IgM and IgD stimuli, were analyzed by immunostaining with antibody against pCblY700, c-Cbl, pLynY396, Lyn and β-actin. ( C ) Proximity ligation assay (PLA) was performed on CLL and normal B cells (Cnt). Rabbit polyclonal anti-Lyn and mouse monoclonal anti-ubiquitin were used as primary Abs. Positive PLA signals are visualized as green fluorescent spots, nuclei are in blue (DAPI). Images are representative of three independent experiments, and were acquired with zoom 3 using 60X/1.35NA oil immersion objective. Z-projections of 15 slices are shown. Scale bar: 5 µm.

Techniques Used: Immunoprecipitation, SDS Page, Stripping Membranes, Western Blot, Immunostaining, Proximity Ligation Assay

Analysis of c-Cbl phosphorylation and its association with PI3K regulatory subunit p85 ( A ) Densitometric ananlysis of c-CblY700/c-CblTot ratio in 24 CLL patients before (Alone) and after 5 minutes of IgM (IgM) and 3 minutes of IgD (IgD). Data obtained were evaluated for their statistical significance with pair Student’s t test ( **** p
Figure Legend Snippet: Analysis of c-Cbl phosphorylation and its association with PI3K regulatory subunit p85 ( A ) Densitometric ananlysis of c-CblY700/c-CblTot ratio in 24 CLL patients before (Alone) and after 5 minutes of IgM (IgM) and 3 minutes of IgD (IgD). Data obtained were evaluated for their statistical significance with pair Student’s t test ( **** p

Techniques Used:

12) Product Images from "Novel immortal human cell lines reveal subpopulations in the nucleus pulposus"

Article Title: Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

Journal: Arthritis Research & Therapy

doi: 10.1186/ar4597

Confirmation of the nucleus pulposus cell phenotype in vitro. (A) Representative phase-contrast images of primary nucleus pulposus (NP) and annular fibrosus (AF) cells from donor 1 (D1) and donor 2 (D2). Passage numbers (P x ) for each donor’s tissue are indicated in parentheses (for example, D1 (P0)). Black bars = 20 μm. (B) Top : Graphed results of gene expression analysis of the chondrocyte markers collagen type I, α1 ( COL1A1 ), and collagen type II, α1 ( COL2A1 ), in primary AF (white bars) and NP (black bars) cell isolates from D1 (P0) and D2 (P1) tissues, respectively. Gene expression was normalized to β-actin (bACT) mRNA levels. Data presented are relative to the AF values. Lower panels : Immunoblot analysis of primary AF and NP cell lysates from two independent donors, D1 (P0) and D3 (P1), respectively, for COL1A1 and COL2A1. bACT was used as a loading control. (C) Representative phase-contrast images of AF and NP cultures from D4 and D5, both at P5. Black bars = 20 μm. (D) Immunoblot analysis of COL1A1, COL2A1 and SRY-box 9 (SOX9) on cell lysates from P5 AF and NP cells. (E) P5 AF and NP cells were stimulated for 7 days with differentiation medium, and cell lysates were analysed for COL1A1, COL2A1 and SOX9. bACT was used as a loading control. (F) Gene expression (mRNA) analysis of six NP markers: keratin 19 ( KRT19 ), Carbonic anhydrase XII ( CA12 ), cluster of differentiation 24 ( CD24 ), Forkhead box F1 ( FoxF1 ), paired box 1 ( Pax1 ) and pleiotrophin ( PTN ) in cultured AF (white bars) and NP cell isolates (black bars) in tissue from three independent donors: D2 (P1), D4 (P5) and D5 (P5). Marker gene expression was normalized to bACT levels. NP data presented are relative to AF values (per patient). a Indicated P -values were obtained by comparing AF and NP values combined for all three donors. Statistical significance was assessed by Student’s t -test. * P
Figure Legend Snippet: Confirmation of the nucleus pulposus cell phenotype in vitro. (A) Representative phase-contrast images of primary nucleus pulposus (NP) and annular fibrosus (AF) cells from donor 1 (D1) and donor 2 (D2). Passage numbers (P x ) for each donor’s tissue are indicated in parentheses (for example, D1 (P0)). Black bars = 20 μm. (B) Top : Graphed results of gene expression analysis of the chondrocyte markers collagen type I, α1 ( COL1A1 ), and collagen type II, α1 ( COL2A1 ), in primary AF (white bars) and NP (black bars) cell isolates from D1 (P0) and D2 (P1) tissues, respectively. Gene expression was normalized to β-actin (bACT) mRNA levels. Data presented are relative to the AF values. Lower panels : Immunoblot analysis of primary AF and NP cell lysates from two independent donors, D1 (P0) and D3 (P1), respectively, for COL1A1 and COL2A1. bACT was used as a loading control. (C) Representative phase-contrast images of AF and NP cultures from D4 and D5, both at P5. Black bars = 20 μm. (D) Immunoblot analysis of COL1A1, COL2A1 and SRY-box 9 (SOX9) on cell lysates from P5 AF and NP cells. (E) P5 AF and NP cells were stimulated for 7 days with differentiation medium, and cell lysates were analysed for COL1A1, COL2A1 and SOX9. bACT was used as a loading control. (F) Gene expression (mRNA) analysis of six NP markers: keratin 19 ( KRT19 ), Carbonic anhydrase XII ( CA12 ), cluster of differentiation 24 ( CD24 ), Forkhead box F1 ( FoxF1 ), paired box 1 ( Pax1 ) and pleiotrophin ( PTN ) in cultured AF (white bars) and NP cell isolates (black bars) in tissue from three independent donors: D2 (P1), D4 (P5) and D5 (P5). Marker gene expression was normalized to bACT levels. NP data presented are relative to AF values (per patient). a Indicated P -values were obtained by comparing AF and NP values combined for all three donors. Statistical significance was assessed by Student’s t -test. * P

Techniques Used: In Vitro, Expressing, Cell Culture, Marker

13) Product Images from "αv Integrins regulate germinal center B cell responses through noncanonical autophagy"

Article Title: αv Integrins regulate germinal center B cell responses through noncanonical autophagy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI99597

Loss of α v affects long-lived antibody responses. ( A ) Serum anti-VLP IgG and IgG2c titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA that were boosted with empty VLP at day 68 and harvested after a further 7 days. ( B ) Frequency of VLP-specific CD38 + IgD lo B cells in control and α v -CD19 mice at day 7 after boost as in A . ( C and D ) Antigen-specific plasma cells enumerated by ELISpot assay on BM cells from control or α v -CD19 mice harvested after immunization and rechallenged with either ( C ) VLPs (2 μg) or ( D ) NP-CG with imiquimod-SE. ( E ) Serum anti-NP IgG, IgG1, and IgG2c titers in control and α v -CD19 mice immunized initially with NP-CG with either LPS or alum and boosted at day 42 with NP-CG (25 μg) alone. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Loss of α v affects long-lived antibody responses. ( A ) Serum anti-VLP IgG and IgG2c titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA that were boosted with empty VLP at day 68 and harvested after a further 7 days. ( B ) Frequency of VLP-specific CD38 + IgD lo B cells in control and α v -CD19 mice at day 7 after boost as in A . ( C and D ) Antigen-specific plasma cells enumerated by ELISpot assay on BM cells from control or α v -CD19 mice harvested after immunization and rechallenged with either ( C ) VLPs (2 μg) or ( D ) NP-CG with imiquimod-SE. ( E ) Serum anti-NP IgG, IgG1, and IgG2c titers in control and α v -CD19 mice immunized initially with NP-CG with either LPS or alum and boosted at day 42 with NP-CG (25 μg) alone. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, MANN-WHITNEY

14) Product Images from "Cytoplasmic myosin exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability"

Article Title: Cytoplasmic myosin exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability

Journal: Leukemia

doi: 10.1038/leu.2015.204

CLL cells form MEACs during apoptosis. ( a ) Flow cytometric analyses of CD19 + CD3 − CLL cells are displayed as contour plots of fluorescence intensity shown on five-log scales with BiExponential transformation. Cells were cultured for 0, 1, 2, or 3 days and stained with rabbit anti-human myosin, FITC-conjugated anti-rabbit IgG, 7AAD and AnnexinV. Representative results from four experiments with seven CLL patient samples are illustrated with two CLL patients, CLL321 and CLL693. IGHV gene and mutation status are labeled. The top row for each patient shows plots of 7AAD and AnnexinV with % of live (7AAD − , AnnexinV − ), early apoptotic (7AAD − , AnnexinV + ), and late apoptotic (7AAD + , AnnexinV + ) cell populations over time. The bottom row for each patient shows AnnexinV and anti-myosin plots with MEAC % from the same experiment. ( b ) Apoptotic CLL693 cells stained with CFSE (green) and anti-myosin (red) were visualized separately by a confocal laser-scanning microscope system (FluoView 300-1X; Olympus) using a PLAN APO 60X/1.4 oil-objective lens with a 1.5X digital zoom and then the images were merged. Three representative CLL MEAC images are shown from multiple images obtained in 8 independent experiments. ( c ) Apoptotic CLL693 cells stained with anti-CD23 (blue) and anti-myosin (red) were visualized by confocal microscopy as in ( b ) except with a 3X digital zoom. ( b–c ) Three representative CLL MEAC images are shown from multiple images obtained in 7 independent experiments.
Figure Legend Snippet: CLL cells form MEACs during apoptosis. ( a ) Flow cytometric analyses of CD19 + CD3 − CLL cells are displayed as contour plots of fluorescence intensity shown on five-log scales with BiExponential transformation. Cells were cultured for 0, 1, 2, or 3 days and stained with rabbit anti-human myosin, FITC-conjugated anti-rabbit IgG, 7AAD and AnnexinV. Representative results from four experiments with seven CLL patient samples are illustrated with two CLL patients, CLL321 and CLL693. IGHV gene and mutation status are labeled. The top row for each patient shows plots of 7AAD and AnnexinV with % of live (7AAD − , AnnexinV − ), early apoptotic (7AAD − , AnnexinV + ), and late apoptotic (7AAD + , AnnexinV + ) cell populations over time. The bottom row for each patient shows AnnexinV and anti-myosin plots with MEAC % from the same experiment. ( b ) Apoptotic CLL693 cells stained with CFSE (green) and anti-myosin (red) were visualized separately by a confocal laser-scanning microscope system (FluoView 300-1X; Olympus) using a PLAN APO 60X/1.4 oil-objective lens with a 1.5X digital zoom and then the images were merged. Three representative CLL MEAC images are shown from multiple images obtained in 8 independent experiments. ( c ) Apoptotic CLL693 cells stained with anti-CD23 (blue) and anti-myosin (red) were visualized by confocal microscopy as in ( b ) except with a 3X digital zoom. ( b–c ) Three representative CLL MEAC images are shown from multiple images obtained in 7 independent experiments.

Techniques Used: Flow Cytometry, Fluorescence, Transformation Assay, Cell Culture, Staining, Mutagenesis, Labeling, Laser-Scanning Microscopy, Confocal Microscopy

Natural IgM antibodies recognize MEACs. ( a ) Flow cytometric analyses of Jurkat cells undergoing spontaneous apoptosis are displayed as contour plots of fluorescence intensity shown on five-log scales with BiExponential transformation. Cells were stained with rabbit anti-myosin and normal human adult serum IgM (Normal IgM), cord blood, or control clonal IgM (OBT1524 or MM021), followed by secondary Abs: PE-conjugated anti-rabbit IgG and FITC-conjugated anti-human IgM. Representative plots of unstained (top left panel), anti-myosin alone (top middle panel), or anti-myosin plus normal human serum (two different lots, 16 experiments), cord blood (6 different samples, 8 experiments), or control clonal IgM stained cells (remaining panels, 8 experiments) are shown. ( b ) After gating on myosin + cells in above plots, histograms of the % of maximum (% of Max) fluorescent intensity for indicated staining (thick blue line) are shown relative to staining with anti-myosin alone (red shaded plot, negative control). ( c ) The mean MFIR was calculated from the geometric mean fluorescence intensity determined in panel B as a ratio over the negative control. MFIR ≥ 1.5 considered positive MEAC binding (dashed red line). 14 Average MFIR with standard error is shown for normal IgM (N=23), cord blood (N=17), OBT1524 (N=3), and MM021 (N=13).
Figure Legend Snippet: Natural IgM antibodies recognize MEACs. ( a ) Flow cytometric analyses of Jurkat cells undergoing spontaneous apoptosis are displayed as contour plots of fluorescence intensity shown on five-log scales with BiExponential transformation. Cells were stained with rabbit anti-myosin and normal human adult serum IgM (Normal IgM), cord blood, or control clonal IgM (OBT1524 or MM021), followed by secondary Abs: PE-conjugated anti-rabbit IgG and FITC-conjugated anti-human IgM. Representative plots of unstained (top left panel), anti-myosin alone (top middle panel), or anti-myosin plus normal human serum (two different lots, 16 experiments), cord blood (6 different samples, 8 experiments), or control clonal IgM stained cells (remaining panels, 8 experiments) are shown. ( b ) After gating on myosin + cells in above plots, histograms of the % of maximum (% of Max) fluorescent intensity for indicated staining (thick blue line) are shown relative to staining with anti-myosin alone (red shaded plot, negative control). ( c ) The mean MFIR was calculated from the geometric mean fluorescence intensity determined in panel B as a ratio over the negative control. MFIR ≥ 1.5 considered positive MEAC binding (dashed red line). 14 Average MFIR with standard error is shown for normal IgM (N=23), cord blood (N=17), OBT1524 (N=3), and MM021 (N=13).

Techniques Used: Flow Cytometry, Fluorescence, Transformation Assay, Staining, Negative Control, Binding Assay

MEACs accumulate over time during both early and late apoptosis. ( a ) Flow cytometric analyses of Jurkat cells are displayed as contour plots of fluorescence intensity shown on five-log scales with BiExponential transformation. Cells were treated with camptothecin for 0, 4, 16, or 24 h to induce apoptosis and stained with rabbit anti-human myosin, FITC-conjugated anti-rabbit IgG, 7AAD and AnnexinV. Representatives of 11 experiments are shown. The top row shows plots of 7AAD and AnnexinV illustrating changes in live (7AAD − , AnnexinV − ), early apoptotic (7AAD − , AnnexinV + ), and late apoptotic (7AAD + , AnnexinV + ) cell populations over time. The bottom row shows AnnexinV and anti-myosin plots for the same experiment. ( b – e ) % of total apoptotic cells (AnnexinV + ) and MEACs were determined by flow cytometry as in ( a ) and plotted over time of incubation with means ± SD shown. ( b ) Summary of camptothecin induced apoptosis and MEAC formation (N=11). ( c ) Summary of spontaneous apoptosis and MEAC formation (N=18). ( d ) Summary of FasL-induced apoptosis and MEAC formation (N=8). ( e ) Summary of anti-Fas induced apoptosis and MEAC formation (N=6). ( f ) Representative example of camptothecin treated Jurkat cells analyzed by flow cytometry for apoptosis stage by AnnexinV and 7AAD staining after 4 h (left) or 24 h (right) of incubation. Early (7AAD − , AnnexinV + ) and late (7AAD + , AnnexinV + ) apoptotic cells were gated and analyzed for MEAC % by level of myosin staining (bottom).
Figure Legend Snippet: MEACs accumulate over time during both early and late apoptosis. ( a ) Flow cytometric analyses of Jurkat cells are displayed as contour plots of fluorescence intensity shown on five-log scales with BiExponential transformation. Cells were treated with camptothecin for 0, 4, 16, or 24 h to induce apoptosis and stained with rabbit anti-human myosin, FITC-conjugated anti-rabbit IgG, 7AAD and AnnexinV. Representatives of 11 experiments are shown. The top row shows plots of 7AAD and AnnexinV illustrating changes in live (7AAD − , AnnexinV − ), early apoptotic (7AAD − , AnnexinV + ), and late apoptotic (7AAD + , AnnexinV + ) cell populations over time. The bottom row shows AnnexinV and anti-myosin plots for the same experiment. ( b – e ) % of total apoptotic cells (AnnexinV + ) and MEACs were determined by flow cytometry as in ( a ) and plotted over time of incubation with means ± SD shown. ( b ) Summary of camptothecin induced apoptosis and MEAC formation (N=11). ( c ) Summary of spontaneous apoptosis and MEAC formation (N=18). ( d ) Summary of FasL-induced apoptosis and MEAC formation (N=8). ( e ) Summary of anti-Fas induced apoptosis and MEAC formation (N=6). ( f ) Representative example of camptothecin treated Jurkat cells analyzed by flow cytometry for apoptosis stage by AnnexinV and 7AAD staining after 4 h (left) or 24 h (right) of incubation. Early (7AAD − , AnnexinV + ) and late (7AAD + , AnnexinV + ) apoptotic cells were gated and analyzed for MEAC % by level of myosin staining (bottom).

Techniques Used: Flow Cytometry, Fluorescence, Transformation Assay, Staining, Cytometry, Incubation

15) Product Images from "Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens"

Article Title: Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens

Journal: The EMBO Journal

doi: 10.15252/embj.201694177

Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding Calcium flux measured by FACScan for splenic B cells isolated from Lyn‐deficient B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified Fab‐PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Data information: Data are representative of at least three independent experiments.
Figure Legend Snippet: Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding Calcium flux measured by FACScan for splenic B cells isolated from Lyn‐deficient B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified Fab‐PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Data information: Data are representative of at least three independent experiments.

Techniques Used: Binding Assay, Isolation, Transgenic Assay, Mouse Assay, Proximity Ligation Assay, Staining

The kinase activity of Lyn is crucial for monovalent antigen‐induced calcium response and BCR opening Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after the stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45‐min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for IgM‐BCR and IgD‐BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments.
Figure Legend Snippet: The kinase activity of Lyn is crucial for monovalent antigen‐induced calcium response and BCR opening Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after the stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45‐min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for IgM‐BCR and IgD‐BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments.

Techniques Used: Activity Assay, Isolation, Transgenic Assay, Mouse Assay, Incubation, Proximity Ligation Assay, Staining

Monovalent antigen binding to IgM‐ BCR induces calcium signalling in a SLP ‐65 dependent manner Proximity between IgM‐BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1‐min stimulation with the indicated reagents, assayed by Fab‐PLA. Results are presented as representative microscopic images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample, and P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Data information: Data are representative of a minimum of three independent experiments.
Figure Legend Snippet: Monovalent antigen binding to IgM‐ BCR induces calcium signalling in a SLP ‐65 dependent manner Proximity between IgM‐BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1‐min stimulation with the indicated reagents, assayed by Fab‐PLA. Results are presented as representative microscopic images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample, and P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Data information: Data are representative of a minimum of three independent experiments.

Techniques Used: Binding Assay, Proximity Ligation Assay, Staining

Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (see Fig EV1 , 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. Representative microscopic images showing Fab‐PLA results measuring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. The Fab‐PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments.
Figure Legend Snippet: Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (see Fig EV1 , 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. Representative microscopic images showing Fab‐PLA results measuring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. The Fab‐PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments.

Techniques Used: Binding Assay, Isolation, Transgenic Assay, Mouse Assay, Proximity Ligation Assay, Staining, Software

16) Product Images from "Disruption of TGF-? signaling in T cells accelerates atherosclerosis"

Article Title: Disruption of TGF-? signaling in T cells accelerates atherosclerosis

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200318607

Lesion composition in 12-week-old E 0 mice with abrogated TGF-β signaling in T cells. Comparison with size-matched lesions of 18-week-old E 0 mice. ( a ) Collagen content (collagen-stained cross-section surface/total lesion surface). ( b ) Area occupied by CD68 + macrophages (CD68-stained surface/total lesion surface). ( c ) I-A + cells per lesion cross-section area. ( d ) CD3 + T cells per lesion cross-section area. ( e ) Representative micrographs showing Masson′s trichrome stain (collagen in green; original magnification ×400), immunohistochemical staining for CD68, I-A, and CD3 (all ×400), and immunohistochemical staining for α-smooth muscle actin (α actin) and VCAM-1 (×200). Lesion contours are outlined in the CD3-stained micrograph.
Figure Legend Snippet: Lesion composition in 12-week-old E 0 mice with abrogated TGF-β signaling in T cells. Comparison with size-matched lesions of 18-week-old E 0 mice. ( a ) Collagen content (collagen-stained cross-section surface/total lesion surface). ( b ) Area occupied by CD68 + macrophages (CD68-stained surface/total lesion surface). ( c ) I-A + cells per lesion cross-section area. ( d ) CD3 + T cells per lesion cross-section area. ( e ) Representative micrographs showing Masson′s trichrome stain (collagen in green; original magnification ×400), immunohistochemical staining for CD68, I-A, and CD3 (all ×400), and immunohistochemical staining for α-smooth muscle actin (α actin) and VCAM-1 (×200). Lesion contours are outlined in the CD3-stained micrograph.

Techniques Used: Mouse Assay, Staining, Immunohistochemistry

17) Product Images from "TRAF3 is required for T cell-mediated immunity and T cell receptor/CD28 signaling 1"

Article Title: TRAF3 is required for T cell-mediated immunity and T cell receptor/CD28 signaling 1

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

doi: 10.4049/jimmunol.1000290

Defective T-dependent IgG1 response in T-TRAF3 −/− mice
Figure Legend Snippet: Defective T-dependent IgG1 response in T-TRAF3 −/− mice

Techniques Used: Mouse Assay

18) Product Images from "Testosterone is an endogenous regulator of BAFF and splenic B cell number"

Article Title: Testosterone is an endogenous regulator of BAFF and splenic B cell number

Journal: Nature Communications

doi: 10.1038/s41467-018-04408-0

Testosterone regulates splenic catecholamine levels. a Noradrenaline and b dopamine concentration in spleens of castrated (ORX; n = 6) and sham-operated ( n = 5) male mice. c Quantification of tyrosine hydroxylase (TH)-stained area in control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) male mice, expressed as percentage of follicle area. n = 7/group. d–f Sections of spleens from control and G-ARKO mice. Turquoise, nerve structures (tyrosine hydroxylase; TH); red, fibroblastic reticular cells (FRCs) and vascular structures (smooth muscle a-actin; SMA). B cells (IgD) are stained white in e and BAFF is stained white in f . Scale bar, 100 μm. g Higher-magnification image of SMA-positive FRC (red) and TH-stained nerve structures (turquoise); asterisk indicates area of co-localization. Scale bar, 20 μm. Bars indicate means; circles represent individual mice. * P
Figure Legend Snippet: Testosterone regulates splenic catecholamine levels. a Noradrenaline and b dopamine concentration in spleens of castrated (ORX; n = 6) and sham-operated ( n = 5) male mice. c Quantification of tyrosine hydroxylase (TH)-stained area in control ( Pgk-Cre + ) and general androgen receptor knockout (G-ARKO) male mice, expressed as percentage of follicle area. n = 7/group. d–f Sections of spleens from control and G-ARKO mice. Turquoise, nerve structures (tyrosine hydroxylase; TH); red, fibroblastic reticular cells (FRCs) and vascular structures (smooth muscle a-actin; SMA). B cells (IgD) are stained white in e and BAFF is stained white in f . Scale bar, 100 μm. g Higher-magnification image of SMA-positive FRC (red) and TH-stained nerve structures (turquoise); asterisk indicates area of co-localization. Scale bar, 20 μm. Bars indicate means; circles represent individual mice. * P

Techniques Used: Concentration Assay, Mouse Assay, Staining, Knock-Out

19) Product Images from "Cytoskeletal adaptivity regulates T cell receptor signaling"

Article Title: Cytoskeletal adaptivity regulates T cell receptor signaling

Journal: Science signaling

doi: 10.1126/scisignal.aah3737

Compared to naïve CD4 + T cells, effector CD4 + T cells are more reactive, form larger immune synapses, and are mechanically softer ( A ) Maximum Ca 2+ flux responses in naïve and effector OT-II T cells interacting with antigen-pulsed APCs. Each dot represents an individual T cell. Maximum fluorescence was calculated as an average over the whole cell. Ca 2+ flux was scaled to the brightness of each cell at time 0. Boxes show means and 95% confidence interval (CI) values. The numbers of Ca 2+ fluxes measured for each group in a single experiment are shown. Results are representative of two independent experiments. ( B ) Immune synapses formed by naïve (top) and effector (bottom) OT-II T cells interacting with antigen-pulsed APCs were imaged by confocal microscopy. Images show a single confocal slice through the middle of the immune synapse. Representative cells that had the median volume of LFA-1 + voxels were chosen. Images are representative of 151 total cells from two independent experiments. ( C ) Effector T cells have a larger volume of LFA-1 + voxels in the immune synapse. The total volume of LFA-1 represents the size of the immune synapse. The average immune synapse size for naïve cells was normalized to 1. Each dot represents an immune synapse between a T cell and an APC. Boxes show means and 95% CI values. The numbers of synapses measured for each group in a single experiment are shown. Results are representative of two independent experiments. ( D ) Elastic moduli derived from nanoindentation measurements for live naïve and effector T cells. Each dot is the average of about five measurements of single T cells. Boxes show means and 95% CI values. The numbers of cells measured for each group in a single experiment are shown. Results are representative of two independent experiments. ( E ) Stiffness mapping of naïve (top) and effector (bottom) T cells. Smoothed density histograms comprising ~160 nanoindentation measurements for each cell are shown. Each shade represents a distinct cell that was measured. The stiffness maps of individual T cells showed a range of elastic moduli from ~100 Pa to 100 kPa, with median stiffness for each cell shown in yellow circles. The median stiffness values for the touches on naïve or effector cells are shown as orange vertical lines. The numbers of cells measured for each group in a single experiment are shown. Results are representative of three independent experiments.
Figure Legend Snippet: Compared to naïve CD4 + T cells, effector CD4 + T cells are more reactive, form larger immune synapses, and are mechanically softer ( A ) Maximum Ca 2+ flux responses in naïve and effector OT-II T cells interacting with antigen-pulsed APCs. Each dot represents an individual T cell. Maximum fluorescence was calculated as an average over the whole cell. Ca 2+ flux was scaled to the brightness of each cell at time 0. Boxes show means and 95% confidence interval (CI) values. The numbers of Ca 2+ fluxes measured for each group in a single experiment are shown. Results are representative of two independent experiments. ( B ) Immune synapses formed by naïve (top) and effector (bottom) OT-II T cells interacting with antigen-pulsed APCs were imaged by confocal microscopy. Images show a single confocal slice through the middle of the immune synapse. Representative cells that had the median volume of LFA-1 + voxels were chosen. Images are representative of 151 total cells from two independent experiments. ( C ) Effector T cells have a larger volume of LFA-1 + voxels in the immune synapse. The total volume of LFA-1 represents the size of the immune synapse. The average immune synapse size for naïve cells was normalized to 1. Each dot represents an immune synapse between a T cell and an APC. Boxes show means and 95% CI values. The numbers of synapses measured for each group in a single experiment are shown. Results are representative of two independent experiments. ( D ) Elastic moduli derived from nanoindentation measurements for live naïve and effector T cells. Each dot is the average of about five measurements of single T cells. Boxes show means and 95% CI values. The numbers of cells measured for each group in a single experiment are shown. Results are representative of two independent experiments. ( E ) Stiffness mapping of naïve (top) and effector (bottom) T cells. Smoothed density histograms comprising ~160 nanoindentation measurements for each cell are shown. Each shade represents a distinct cell that was measured. The stiffness maps of individual T cells showed a range of elastic moduli from ~100 Pa to 100 kPa, with median stiffness for each cell shown in yellow circles. The median stiffness values for the touches on naïve or effector cells are shown as orange vertical lines. The numbers of cells measured for each group in a single experiment are shown. Results are representative of three independent experiments.

Techniques Used: Fluorescence, Confocal Microscopy, Derivative Assay

20) Product Images from "Autocrine transforming growth factor ? signaling regulates extracellular signal-regulated kinase 1/2 phosphorylation via modulation of protein phosphatase 2A expression in scleroderma fibroblasts"

Article Title: Autocrine transforming growth factor ? signaling regulates extracellular signal-regulated kinase 1/2 phosphorylation via modulation of protein phosphatase 2A expression in scleroderma fibroblasts

Journal: Fibrogenesis & Tissue Repair

doi: 10.1186/1755-1536-3-25

Autocrine transforming growth factor (TGF)β signaling regulates protein phosphatase 2A (PP2A) expression in scleroderma (SSc) fibroblasts . (a) Normal dermal fibroblasts were pretreated with recombinant transforming growth factor β soluble receptor II (SRII) (10 ng/ml) for 1 h and then treated with 5 ng/ml of TGFβ for 24 h. Western blot analysis was used to determine PP2A levels. (b) Normal and (c) SSc dermal fibroblasts were treated with SRII for 24 h after serum deprivation. Cells were collected and total protein levels of PP2A catalytic subunit were analyzed by western blot. In SSc cells, cell lysates were also analyzed for phospho-extracellular signal-regulated kinase (ERK)1/2 and type 1 collagen. β Actin was used as a loading control. (d) Bar graph shows quantification of PP2A protein expression after treatment of SSc fibroblasts with SRII; * P
Figure Legend Snippet: Autocrine transforming growth factor (TGF)β signaling regulates protein phosphatase 2A (PP2A) expression in scleroderma (SSc) fibroblasts . (a) Normal dermal fibroblasts were pretreated with recombinant transforming growth factor β soluble receptor II (SRII) (10 ng/ml) for 1 h and then treated with 5 ng/ml of TGFβ for 24 h. Western blot analysis was used to determine PP2A levels. (b) Normal and (c) SSc dermal fibroblasts were treated with SRII for 24 h after serum deprivation. Cells were collected and total protein levels of PP2A catalytic subunit were analyzed by western blot. In SSc cells, cell lysates were also analyzed for phospho-extracellular signal-regulated kinase (ERK)1/2 and type 1 collagen. β Actin was used as a loading control. (d) Bar graph shows quantification of PP2A protein expression after treatment of SSc fibroblasts with SRII; * P

Techniques Used: Expressing, Recombinant, Western Blot

21) Product Images from "Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins"

Article Title: Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

Journal: Nature Communications

doi: 10.1038/ncomms12754

The wt NS1B protein, but not the 67 mutant NS1B protein, binds and sequesters ISG15 conjugates in IFN-β-pretreated influenza B virus-infected cells. ( a ) Schematic representation of the two IFN-β-inducible, S-3F-tagged ISG15 sequences inserted into A549 cells. ( b ) S-3F-ISG15 A549 cells were either untreated or treated with 1,000 IU ml −1 of human IFN-β for 24 h. Cell extracts were analysed by immunoblots probed with Flag Ab. ( c ) S-3F-ISG15 A549 cells were transfected with control (ctrl) siRNA or E1(UbelL)-specific siRNA for 24 h, followed by treatment with 1,000 IU ml −1 of human IFN-β for 12 h. Cells were then infected with 10 p.f.u. per cell of either wt or 67 mutant virus for 18 h. Cell extracts were analysed by immunoblots probed with Flag Ab. ( d ) S-3 F-ISG15 mut or wt cells were treated with 1,000 IU ml −1 of human IFN-β for 16 h, and then infected with 10 p.f.u. per cell of wt virus for 24 h. Denatured cell extracts were subject to double-affinity purification using anti-Flag M2 agarose and S-protein agarose. The purified proteins were detected by silver staining after SDS page. ( e ) The cell extracts that were analysed in lanes 1 and 3 of panel c were instead immunoprecipitated with NS1B Ab, and the immunoprecipitates were then analysed by immunoblots probed with Flag Ab and NS1B Ab.
Figure Legend Snippet: The wt NS1B protein, but not the 67 mutant NS1B protein, binds and sequesters ISG15 conjugates in IFN-β-pretreated influenza B virus-infected cells. ( a ) Schematic representation of the two IFN-β-inducible, S-3F-tagged ISG15 sequences inserted into A549 cells. ( b ) S-3F-ISG15 A549 cells were either untreated or treated with 1,000 IU ml −1 of human IFN-β for 24 h. Cell extracts were analysed by immunoblots probed with Flag Ab. ( c ) S-3F-ISG15 A549 cells were transfected with control (ctrl) siRNA or E1(UbelL)-specific siRNA for 24 h, followed by treatment with 1,000 IU ml −1 of human IFN-β for 12 h. Cells were then infected with 10 p.f.u. per cell of either wt or 67 mutant virus for 18 h. Cell extracts were analysed by immunoblots probed with Flag Ab. ( d ) S-3 F-ISG15 mut or wt cells were treated with 1,000 IU ml −1 of human IFN-β for 16 h, and then infected with 10 p.f.u. per cell of wt virus for 24 h. Denatured cell extracts were subject to double-affinity purification using anti-Flag M2 agarose and S-protein agarose. The purified proteins were detected by silver staining after SDS page. ( e ) The cell extracts that were analysed in lanes 1 and 3 of panel c were instead immunoprecipitated with NS1B Ab, and the immunoprecipitates were then analysed by immunoblots probed with Flag Ab and NS1B Ab.

Techniques Used: Mutagenesis, Infection, Western Blot, Transfection, Affinity Purification, Purification, Silver Staining, SDS Page, Immunoprecipitation

The NS1B protein does not inhibit IFN-β-induced ISGylation in influenza B virus-infected cells. ( a ) Multiple cycle growth curves of wt and 67 mutant virus in A549 cells with or without IFN-β pretreatment. Where indicated, A549 cells were treated with 1,000 IU ml −1 of human IFN-β for 18 h before virus infection. Cells were infected with 0.1 p.f.u. per cell of wt virus or 67 mutant virus. Error bars show the s.d. of triplicate assays of virus titers at the indicated times of infection determined by plaque assays in MDCK cells. ( b ) The effect of siRNA knockdown of ISG15 on the multiple cycle growth of wt and 67 mutant virus in IFN-β pretreated A549 cells. Where indicated, ISG15-specific siRNA was transfected into cells for 24 h before IFN-β treatment. The efficiency of the ISG15-specific siRNA in inhibiting ISGylation is shown in Fig. 4a , lanes 5 and 6. Error bars show the s.d. of triplicate assays of virus titers at the indicated times of infection. See also ( Supplementary Fig. 1a,b ). ( c ) The effect of wt and 67 mutant virus on IFN-β-induced ISGylation. A549 cells were treated with 1,000 IU ml −1 of human IFN-β for 12 h or were not treated with IFN. Cells were then either mock infected, or infected with 5 p.f.u. per cell of wt or 67 mutant virus. Cell extracts isolated at indicated times after infection or mock infection were analysed by immunoblots probed with the indicated Abs.
Figure Legend Snippet: The NS1B protein does not inhibit IFN-β-induced ISGylation in influenza B virus-infected cells. ( a ) Multiple cycle growth curves of wt and 67 mutant virus in A549 cells with or without IFN-β pretreatment. Where indicated, A549 cells were treated with 1,000 IU ml −1 of human IFN-β for 18 h before virus infection. Cells were infected with 0.1 p.f.u. per cell of wt virus or 67 mutant virus. Error bars show the s.d. of triplicate assays of virus titers at the indicated times of infection determined by plaque assays in MDCK cells. ( b ) The effect of siRNA knockdown of ISG15 on the multiple cycle growth of wt and 67 mutant virus in IFN-β pretreated A549 cells. Where indicated, ISG15-specific siRNA was transfected into cells for 24 h before IFN-β treatment. The efficiency of the ISG15-specific siRNA in inhibiting ISGylation is shown in Fig. 4a , lanes 5 and 6. Error bars show the s.d. of triplicate assays of virus titers at the indicated times of infection. See also ( Supplementary Fig. 1a,b ). ( c ) The effect of wt and 67 mutant virus on IFN-β-induced ISGylation. A549 cells were treated with 1,000 IU ml −1 of human IFN-β for 12 h or were not treated with IFN. Cells were then either mock infected, or infected with 5 p.f.u. per cell of wt or 67 mutant virus. Cell extracts isolated at indicated times after infection or mock infection were analysed by immunoblots probed with the indicated Abs.

Techniques Used: Infection, Mutagenesis, Transfection, Isolation, Western Blot

The major target of ISGylation in influenza B virus-infected cells is NP. ( a ) Colloidal blue-stained gel of ISG15 conjugates purified by double-affinity selection from S-3F-ISG15GG A549 cells which were pretreated with IFN-β and then infected with wt virus for 24 h. The four bands that were analysed by mass spectrometry and the ISG15 conjugation sites that were identified in these four bands are shown. See also Supplementary Table 1 . ( b ) Location of the ISG15-modified lysines (Ks) mapped onto the structure of influenza B virus NP (PDB accession code 3TJ0). ( c ) S-3F-ISG15GG A549 cells were pretreated with IFN-β and then infected with 10 p.f.u. per cell of wt or 67 mutant virus for 24 h. Cell extracts and ISG15 conjugates purified from these extracts by double-affinity selection were analysed by immunoblots probed with NP Ab.
Figure Legend Snippet: The major target of ISGylation in influenza B virus-infected cells is NP. ( a ) Colloidal blue-stained gel of ISG15 conjugates purified by double-affinity selection from S-3F-ISG15GG A549 cells which were pretreated with IFN-β and then infected with wt virus for 24 h. The four bands that were analysed by mass spectrometry and the ISG15 conjugation sites that were identified in these four bands are shown. See also Supplementary Table 1 . ( b ) Location of the ISG15-modified lysines (Ks) mapped onto the structure of influenza B virus NP (PDB accession code 3TJ0). ( c ) S-3F-ISG15GG A549 cells were pretreated with IFN-β and then infected with 10 p.f.u. per cell of wt or 67 mutant virus for 24 h. Cell extracts and ISG15 conjugates purified from these extracts by double-affinity selection were analysed by immunoblots probed with NP Ab.

Techniques Used: Infection, Staining, Purification, Selection, Mass Spectrometry, Conjugation Assay, Modification, Mutagenesis, Western Blot

22) Product Images from "Activation of Bone Morphogenetic Protein 4 Signaling Leads to Glomerulosclerosis That Mimics Diabetic Nephropathy *"

Article Title: Activation of Bone Morphogenetic Protein 4 Signaling Leads to Glomerulosclerosis That Mimics Diabetic Nephropathy *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.179382

AGE and Bmp4 showed increased ECM proteins in mouse mesangial cells. A, expression of Bmp4 and Col4 detected by immunoblot after treatment with AGE (1 or 10 μg/cm 2 ) and BSA (1 or 10 μg/cm 2 ) for 48 h. Equivalent protein loading was confirmed
Figure Legend Snippet: AGE and Bmp4 showed increased ECM proteins in mouse mesangial cells. A, expression of Bmp4 and Col4 detected by immunoblot after treatment with AGE (1 or 10 μg/cm 2 ) and BSA (1 or 10 μg/cm 2 ) for 48 h. Equivalent protein loading was confirmed

Techniques Used: Expressing

Modulation of Bmp4 signaling. A, expression of Bmp4 and Col4 after treatment with AGE, detected by immunoblot with Bmp4 siRNA ( siBmp4 ) or scrambled siRNA (scramble) for 48 h. B, phosphorylation of Smad1/5/8 and expressions of Col4 after stimulation by
Figure Legend Snippet: Modulation of Bmp4 signaling. A, expression of Bmp4 and Col4 after treatment with AGE, detected by immunoblot with Bmp4 siRNA ( siBmp4 ) or scrambled siRNA (scramble) for 48 h. B, phosphorylation of Smad1/5/8 and expressions of Col4 after stimulation by

Techniques Used: Expressing

23) Product Images from "Effect of cancer-associated mutations in the PlexinB1 gene"

Article Title: Effect of cancer-associated mutations in the PlexinB1 gene

Journal: Molecular Cancer

doi: 10.1186/1476-4598-11-11

Mutation of plexinB1 enhances RhoD binding . A).GST-cyto-plexinB1 (WT and mutant) fusion proteins were used in GST pulldown assays with lysates of HEK293 cells expressing constitutively active (G26V) or dominant negative (T31K) RhoD-myc. B). Input of RhoD(G26V)-myc, RhoD(T31K)-myc and WT and mutant GST-cyto-plexinB1 fusion proteins. C). Expression of RhoDGTP decreases cell collapse. COS-7 cells co-transfected with plexinB1, Rnd and RhoD(G26V)-myc, or RhoD(T31K)-myc were stimulated with sema4D for 5mins, fixed and stained for plexinB1 (FITC) and myc (TRITC) and the % cell collapse scored. i).% cell collapse +/- SE, * p
Figure Legend Snippet: Mutation of plexinB1 enhances RhoD binding . A).GST-cyto-plexinB1 (WT and mutant) fusion proteins were used in GST pulldown assays with lysates of HEK293 cells expressing constitutively active (G26V) or dominant negative (T31K) RhoD-myc. B). Input of RhoD(G26V)-myc, RhoD(T31K)-myc and WT and mutant GST-cyto-plexinB1 fusion proteins. C). Expression of RhoDGTP decreases cell collapse. COS-7 cells co-transfected with plexinB1, Rnd and RhoD(G26V)-myc, or RhoD(T31K)-myc were stimulated with sema4D for 5mins, fixed and stained for plexinB1 (FITC) and myc (TRITC) and the % cell collapse scored. i).% cell collapse +/- SE, * p

Techniques Used: Mutagenesis, Binding Assay, Expressing, Dominant Negative Mutation, Transfection, Staining

24) Product Images from "Disruption of SEMA4D ameliorates platelet hypersensitivity in dyslipidemia and confers protection against the development of atherosclerosis"

Article Title: Disruption of SEMA4D ameliorates platelet hypersensitivity in dyslipidemia and confers protection against the development of atherosclerosis

Journal: Arteriosclerosis, thrombosis, and vascular biology

doi: 10.1161/ATVBAHA.109.185405

Loss of sema4D expression reduces atherosclerotic lesion size (A) Aortas after 6 months on the high fat diet showing lipid-rich deposits. (B,C,D) Analysis of lesion size. (E) Sections from the aortic root. Markers: CD4 and CD8, T-cells; CD22, B-cells; CD11b, monocytes, macrophage and neutrophils; smooth muscle actin (αSMA), smooth muscle cells and fibrotic caps. No differences were observed.
Figure Legend Snippet: Loss of sema4D expression reduces atherosclerotic lesion size (A) Aortas after 6 months on the high fat diet showing lipid-rich deposits. (B,C,D) Analysis of lesion size. (E) Sections from the aortic root. Markers: CD4 and CD8, T-cells; CD22, B-cells; CD11b, monocytes, macrophage and neutrophils; smooth muscle actin (αSMA), smooth muscle cells and fibrotic caps. No differences were observed.

Techniques Used: Expressing

25) Product Images from "Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies *"

Article Title: Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.320481

CEACAM16 is expressed in Deiters cells. Expression of CEACAM16 was analyzed in the organ of Corti of adult (P49) Ceacam16 +/+ ( A–F , G , and H ) and 44-week-old Ceacam16 −/− mice ( A , inset ) by immunofluorescence using the monoclonal anti-CEACAM16 antibody 9D5. CEACAM16 ( red ) was observed in the OHC-supporting Deiters cells ( A , C , D , F , and H ), which were identified by the potassium channel protein anti-KIR4.1 antibody ( green ) but not in OHCs, which were specifically identified by anti-prestin antibodies ( green ). The shape of OHCs was outlined by ( C ). No CEACAM16 staining could be detected in Ceacam16 −/− mice ( A , inset ). Note the staining for CEACAM16 in both the cell body and projections of the Deiters cells ( open arrows in A and D ), which extend to the reticular lamina ( arrows in C ), the stiff cover of the organ of Corti. The locations of the Deiters cell nuclei are indicated by full arrows ( B and D ). Nuclei were stained with DAPI ( blue ). Furthermore, staining for CEACAM16 was observed in the supranuclear (neck) region of IHCs (outlined by the stippled line ) and in pillar cells ( G and H ). Pillar cells were identified by staining with anti-KIR4.1 antibody. A schematic representation of OHCs and Deiters cells is shown in I . Magnification bars , 10 μm. BM , basilar membrane; DC , Deiters cell; DP , Deiters cell projection; PC , pillar cell; RL , reticular lamina; SC , stereocilia.
Figure Legend Snippet: CEACAM16 is expressed in Deiters cells. Expression of CEACAM16 was analyzed in the organ of Corti of adult (P49) Ceacam16 +/+ ( A–F , G , and H ) and 44-week-old Ceacam16 −/− mice ( A , inset ) by immunofluorescence using the monoclonal anti-CEACAM16 antibody 9D5. CEACAM16 ( red ) was observed in the OHC-supporting Deiters cells ( A , C , D , F , and H ), which were identified by the potassium channel protein anti-KIR4.1 antibody ( green ) but not in OHCs, which were specifically identified by anti-prestin antibodies ( green ). The shape of OHCs was outlined by ( C ). No CEACAM16 staining could be detected in Ceacam16 −/− mice ( A , inset ). Note the staining for CEACAM16 in both the cell body and projections of the Deiters cells ( open arrows in A and D ), which extend to the reticular lamina ( arrows in C ), the stiff cover of the organ of Corti. The locations of the Deiters cell nuclei are indicated by full arrows ( B and D ). Nuclei were stained with DAPI ( blue ). Furthermore, staining for CEACAM16 was observed in the supranuclear (neck) region of IHCs (outlined by the stippled line ) and in pillar cells ( G and H ). Pillar cells were identified by staining with anti-KIR4.1 antibody. A schematic representation of OHCs and Deiters cells is shown in I . Magnification bars , 10 μm. BM , basilar membrane; DC , Deiters cell; DP , Deiters cell projection; PC , pillar cell; RL , reticular lamina; SC , stereocilia.

Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining

26) Product Images from "Isolation of murine lung endothelial cells"

Article Title: Isolation of murine lung endothelial cells

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.90613.2008

Murine ECs isolated by magnetic bead sorting. Shown are ECs isolated by magnetic bead sorting with ICAM-2 antibody that were immunofluorescently stained for VE-cadherin to identify them and with DAPI to identify their nuclei. The magnetic beads appear
Figure Legend Snippet: Murine ECs isolated by magnetic bead sorting. Shown are ECs isolated by magnetic bead sorting with ICAM-2 antibody that were immunofluorescently stained for VE-cadherin to identify them and with DAPI to identify their nuclei. The magnetic beads appear

Techniques Used: Isolation, Staining, Magnetic Beads

Cell morphology in suspensions of collagenase-digested lung. Cell suspensions of collagenase-digested lungs were plated, fixed, and stained immunofluorescently for ICAM-2, an endothelial cell marker, and with DAPI to identify cell nuclei. A : observed
Figure Legend Snippet: Cell morphology in suspensions of collagenase-digested lung. Cell suspensions of collagenase-digested lungs were plated, fixed, and stained immunofluorescently for ICAM-2, an endothelial cell marker, and with DAPI to identify cell nuclei. A : observed

Techniques Used: Staining, Marker

27) Product Images from "αv Integrins regulate germinal center B cell responses through noncanonical autophagy"

Article Title: αv Integrins regulate germinal center B cell responses through noncanonical autophagy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI99597

Loss of α v affects long-lived antibody responses. ( A ) Serum anti-VLP IgG and IgG2c titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA that were boosted with empty VLP at day 68 and harvested after a further 7 days. ( B ) Frequency of VLP-specific CD38 + IgD lo B cells in control and α v -CD19 mice at day 7 after boost as in A . ( C and D ) Antigen-specific plasma cells enumerated by ELISpot assay on BM cells from control or α v -CD19 mice harvested after immunization and rechallenged with either ( C ) VLPs (2 μg) or ( D ) NP-CG with imiquimod-SE. ( E ) Serum anti-NP IgG, IgG1, and IgG2c titers in control and α v -CD19 mice immunized initially with NP-CG with either LPS or alum and boosted at day 42 with NP-CG (25 μg) alone. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Loss of α v affects long-lived antibody responses. ( A ) Serum anti-VLP IgG and IgG2c titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA that were boosted with empty VLP at day 68 and harvested after a further 7 days. ( B ) Frequency of VLP-specific CD38 + IgD lo B cells in control and α v -CD19 mice at day 7 after boost as in A . ( C and D ) Antigen-specific plasma cells enumerated by ELISpot assay on BM cells from control or α v -CD19 mice harvested after immunization and rechallenged with either ( C ) VLPs (2 μg) or ( D ) NP-CG with imiquimod-SE. ( E ) Serum anti-NP IgG, IgG1, and IgG2c titers in control and α v -CD19 mice immunized initially with NP-CG with either LPS or alum and boosted at day 42 with NP-CG (25 μg) alone. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, MANN-WHITNEY

28) Product Images from "αv Integrins regulate germinal center B cell responses through noncanonical autophagy"

Article Title: αv Integrins regulate germinal center B cell responses through noncanonical autophagy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI99597

Loss of α v affects long-lived antibody responses. ( A ) Serum anti-VLP IgG and IgG2c titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA that were boosted with empty VLP at day 68 and harvested after a further 7 days. ( B ) Frequency of VLP-specific CD38 + IgD lo B cells in control and α v -CD19 mice at day 7 after boost as in A . ( C and D ) Antigen-specific plasma cells enumerated by ELISpot assay on BM cells from control or α v -CD19 mice harvested after immunization and rechallenged with either ( C ) VLPs (2 μg) or ( D ) NP-CG with imiquimod-SE. ( E ) Serum anti-NP IgG, IgG1, and IgG2c titers in control and α v -CD19 mice immunized initially with NP-CG with either LPS or alum and boosted at day 42 with NP-CG (25 μg) alone. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Loss of α v affects long-lived antibody responses. ( A ) Serum anti-VLP IgG and IgG2c titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA that were boosted with empty VLP at day 68 and harvested after a further 7 days. ( B ) Frequency of VLP-specific CD38 + IgD lo B cells in control and α v -CD19 mice at day 7 after boost as in A . ( C and D ) Antigen-specific plasma cells enumerated by ELISpot assay on BM cells from control or α v -CD19 mice harvested after immunization and rechallenged with either ( C ) VLPs (2 μg) or ( D ) NP-CG with imiquimod-SE. ( E ) Serum anti-NP IgG, IgG1, and IgG2c titers in control and α v -CD19 mice immunized initially with NP-CG with either LPS or alum and boosted at day 42 with NP-CG (25 μg) alone. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Deletion of α v enhances antibody response to influenza virus. ( A and B ) Serum anti-PR/8 IgG titers in control and α v -CD19 mice immunized with 10 μg of inactivated H1N1 PR/8 ( A ) and ( B ) boosted at day 57 with 5 μg inactivated PR/8. ( C ) PR/8-specific plasma cells enumerated by ELISpot in BM cells from control (Con) and α v -CD19 mice harvested at day 7 after boost. ( D ) HAI activity in sera from control and α v -CD19 mice at day 21 after PR/8 immunization. ( E and F ) Serum antibody titers against HA from H1N1 PR/8 ( E ) or H1N1 Cal/09 ( F ) in control and α v -CD19 mice at day 7 after boost with inactivated PR/8. ( G ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer. ( H and I ) Serum antibody titers against HA from PR/8 ( H ) or Cal/09 ( I ) in control and α v -CD19 at day 51 after immunization with inactivated PR/8 (10 μg) in imiquimod-SE (10 μg). ( J ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer for mice immunized with PR/8 in imiquimod-SE. ( K ) Survival of control and α v -CD19 mice following intranasal infection with PR/8 ( n ≥ 5 mice/group). ( L ) Anti-PR8 HA titers from surviving mice at day 7 after infection. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test for antibody titers or Mantel-Cox test for survival curves).* P
Figure Legend Snippet: Deletion of α v enhances antibody response to influenza virus. ( A and B ) Serum anti-PR/8 IgG titers in control and α v -CD19 mice immunized with 10 μg of inactivated H1N1 PR/8 ( A ) and ( B ) boosted at day 57 with 5 μg inactivated PR/8. ( C ) PR/8-specific plasma cells enumerated by ELISpot in BM cells from control (Con) and α v -CD19 mice harvested at day 7 after boost. ( D ) HAI activity in sera from control and α v -CD19 mice at day 21 after PR/8 immunization. ( E and F ) Serum antibody titers against HA from H1N1 PR/8 ( E ) or H1N1 Cal/09 ( F ) in control and α v -CD19 mice at day 7 after boost with inactivated PR/8. ( G ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer. ( H and I ) Serum antibody titers against HA from PR/8 ( H ) or Cal/09 ( I ) in control and α v -CD19 at day 51 after immunization with inactivated PR/8 (10 μg) in imiquimod-SE (10 μg). ( J ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer for mice immunized with PR/8 in imiquimod-SE. ( K ) Survival of control and α v -CD19 mice following intranasal infection with PR/8 ( n ≥ 5 mice/group). ( L ) Anti-PR8 HA titers from surviving mice at day 7 after infection. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test for antibody titers or Mantel-Cox test for survival curves).* P

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Activity Assay, Infection, MANN-WHITNEY

Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P
Figure Legend Snippet: Increased antibody production in α v -CD19 mice. ( A – B ) Serum anti-VLP antibody titers in control and α v -CD19 mice immunized with 2 μg VLPs containing ssRNA measured 14 days after immunization ( A ) or over a time course from preimmunization (pre) to 35 days ( B ). VLP-specific Abs were not detected (ND) in preimmunization bleeds. ( C ) Serum anti-NP IgM, IgG, IgG1, and Ig2c titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with TLR7 ligand imiquimod-SE (10 μg). ( D ) Serum anti-NP IgG1 and IgG2c antibody titers in control and α v -CD19 mice immunized with NP-CG (50 μg) combined with LPS (5 μg). All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test). * P

Techniques Used: Mouse Assay, MANN-WHITNEY

29) Product Images from "Chronic Allergic Inflammation Causes Vascular Remodeling and Pulmonary Hypertension in Bmpr2 Hypomorph and Wild-Type Mice"

Article Title: Chronic Allergic Inflammation Causes Vascular Remodeling and Pulmonary Hypertension in Bmpr2 Hypomorph and Wild-Type Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032468

Allergic sensitization in HDM exposed mice. Levels of HDM specific IgG1 and IgE were increased in both WT and Bmpr2 ΔE2 mice after 7 weeks (top panels) and 20 weeks (bottom panels) of HDM exposure. At 7 weeks, HDM specific IgG1 levels in WT mice were slightly higher compared to Bmpr2 ΔE2 mice (n = 4–12 mice/group). *P
Figure Legend Snippet: Allergic sensitization in HDM exposed mice. Levels of HDM specific IgG1 and IgE were increased in both WT and Bmpr2 ΔE2 mice after 7 weeks (top panels) and 20 weeks (bottom panels) of HDM exposure. At 7 weeks, HDM specific IgG1 levels in WT mice were slightly higher compared to Bmpr2 ΔE2 mice (n = 4–12 mice/group). *P

Techniques Used: Mouse Assay

30) Product Images from "Myofibroblast contraction activates latent TGF-?1 from the extracellular matrix"

Article Title: Myofibroblast contraction activates latent TGF-?1 from the extracellular matrix

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200704042

Model of Mf contraction-mediated TGF-β1 activation. The high contractile activity generated by α-SMA in stress fibers is transmitted at sites of integrins binding to RGD sites in the LAP protein as part of the LLC, which also includes TGF-β1 and LTBP-1. (A) When the LLC is anchored in a comparably stiff ECM, cell-mediated stress can induce allosteric changes in LTBP-1 and/or LAP conformation, leading to liberation of TGF-β1; such activated TGF-β1 possibly feeds back by binding to its receptor, which is located close by in the activating cell. (B) In the context of compliant ECM, the LLC is dragged toward the pulling cell but because of the lack of mechanical resistance, no conformation change occurs and TGF-β1 remains latent. Likewise, inhibition of high cell contraction and interaction of integrins with the LLC block mechanical activation of latent TGF-β1.
Figure Legend Snippet: Model of Mf contraction-mediated TGF-β1 activation. The high contractile activity generated by α-SMA in stress fibers is transmitted at sites of integrins binding to RGD sites in the LAP protein as part of the LLC, which also includes TGF-β1 and LTBP-1. (A) When the LLC is anchored in a comparably stiff ECM, cell-mediated stress can induce allosteric changes in LTBP-1 and/or LAP conformation, leading to liberation of TGF-β1; such activated TGF-β1 possibly feeds back by binding to its receptor, which is located close by in the activating cell. (B) In the context of compliant ECM, the LLC is dragged toward the pulling cell but because of the lack of mechanical resistance, no conformation change occurs and TGF-β1 remains latent. Likewise, inhibition of high cell contraction and interaction of integrins with the LLC block mechanical activation of latent TGF-β1.

Techniques Used: Activation Assay, Activity Assay, Generated, Binding Assay, Inhibition, Blocking Assay

31) Product Images from "The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding"

Article Title: The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding

Journal: Veterinary Research

doi: 10.1186/1297-9716-43-61

Identification of recombinant viruses by RFLP and Western blotting. ( a ) Purified DNA from EHV-1, EHV-1gH4, EHV-1gH4Kan and EHV-1gH S440A (left panel) as well as EHV-4, EHV-4gH1Kan and EHV-4gH1 (right panel) were digested with Sal I and Sac II. Fragments in the mutants that appeared as a consequence of the deletion or insertion of gH sequence are marked by arrows. ( b ) Amino acid sequence alignment of EHV-1 and EHV-4 gHs’ N-terminal part. The alignment begins with aa49 to aa 327 (H1 domain homologue, that binds gL, in HSV-2) [ 20 ]. ( c ) Parental and mutant viruses as well as complementing RK13/gH1 cells express gH at similar levels. Cell lysates were prepared either from infected FHK cells or from RK13/gH1 and proteins were separated by SDS-10%-PAGE. The blots were incubated with EHV-1 polyclonal anti-gH antibody and detected with anti-rabbit IgG peroxidase conjugate. For RK13/gH1 cells (left panel), EHV-1 and related mutants (middle panel), and EHV-4 and related mutants (right panel), two bands of approximately 125 and 115kD were detected that are not present in mock-infected cells. β-actin was used as a loading control.
Figure Legend Snippet: Identification of recombinant viruses by RFLP and Western blotting. ( a ) Purified DNA from EHV-1, EHV-1gH4, EHV-1gH4Kan and EHV-1gH S440A (left panel) as well as EHV-4, EHV-4gH1Kan and EHV-4gH1 (right panel) were digested with Sal I and Sac II. Fragments in the mutants that appeared as a consequence of the deletion or insertion of gH sequence are marked by arrows. ( b ) Amino acid sequence alignment of EHV-1 and EHV-4 gHs’ N-terminal part. The alignment begins with aa49 to aa 327 (H1 domain homologue, that binds gL, in HSV-2) [ 20 ]. ( c ) Parental and mutant viruses as well as complementing RK13/gH1 cells express gH at similar levels. Cell lysates were prepared either from infected FHK cells or from RK13/gH1 and proteins were separated by SDS-10%-PAGE. The blots were incubated with EHV-1 polyclonal anti-gH antibody and detected with anti-rabbit IgG peroxidase conjugate. For RK13/gH1 cells (left panel), EHV-1 and related mutants (middle panel), and EHV-4 and related mutants (right panel), two bands of approximately 125 and 115kD were detected that are not present in mock-infected cells. β-actin was used as a loading control.

Techniques Used: Recombinant, Western Blot, Purification, Sequencing, Mutagenesis, Infection, Polyacrylamide Gel Electrophoresis, Incubation

32) Product Images from "One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism"

Article Title: One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism

Journal: ACS Chemical Biology

doi: 10.1021/cb500462t

Toxoplasma gondii sortagging. (a) Toxoplasma gondii tachyzoites were incubated with 500 μM TAMRA-LPETG and 20 μM sortase A for 15 min. Parasites were then washed and incubated with human foreskin fibroblasts. Images show the juxtaposition of bright and fluorescent fields. Yellow arrow: intracellular parasite. White arrows: invading parasite. Scale bar: 10 μm. (b) Toxoplasma gondii tachyzoites were incubated with or without 50 μM enhancer- or VHH7-LPETG and 20 μM sortase A. After 20 min, biotin-LPETG was added for 15 min. Parasites were then washed and incubated with red-cell depleted splenocytes for 1 h at a multiplicity of infection of 5. Cells were then washed and stained with a CD19-specific antibody and fluorescently labeled streptavidin. The histogram shows the percentage of sortagged Toxoplasma gondii ( T.g. ) positive cells within CD19 negative or positive populations. Error bars: standard deviation ( n = 3). (c) Purified B cells from WT or class II MHC knock out (k.o.) mice were incubated together with control T . gondii or T. gondii sortagged with enhancer or VHH7 at a multiplicity of infection of 5. Fifteen hours after infection, cell lysis was measured and normalized to uninfected (0%) and detergent-lysed B cells (100%). Error bars: standard deviation ( n = 3). ** p
Figure Legend Snippet: Toxoplasma gondii sortagging. (a) Toxoplasma gondii tachyzoites were incubated with 500 μM TAMRA-LPETG and 20 μM sortase A for 15 min. Parasites were then washed and incubated with human foreskin fibroblasts. Images show the juxtaposition of bright and fluorescent fields. Yellow arrow: intracellular parasite. White arrows: invading parasite. Scale bar: 10 μm. (b) Toxoplasma gondii tachyzoites were incubated with or without 50 μM enhancer- or VHH7-LPETG and 20 μM sortase A. After 20 min, biotin-LPETG was added for 15 min. Parasites were then washed and incubated with red-cell depleted splenocytes for 1 h at a multiplicity of infection of 5. Cells were then washed and stained with a CD19-specific antibody and fluorescently labeled streptavidin. The histogram shows the percentage of sortagged Toxoplasma gondii ( T.g. ) positive cells within CD19 negative or positive populations. Error bars: standard deviation ( n = 3). (c) Purified B cells from WT or class II MHC knock out (k.o.) mice were incubated together with control T . gondii or T. gondii sortagged with enhancer or VHH7 at a multiplicity of infection of 5. Fifteen hours after infection, cell lysis was measured and normalized to uninfected (0%) and detergent-lysed B cells (100%). Error bars: standard deviation ( n = 3). ** p

Techniques Used: Incubation, Infection, Staining, Labeling, Standard Deviation, Purification, Knock-Out, Mouse Assay, Lysis

Sortagging of genetically unmanipulated cells. (a) Conjugation of G (n) -probe to the C-terminus of an LPETG-tagged protein using sortase A. (b) Conjugation of probe-LPETG to the N-terminus of a G (n) -tagged protein using sortase A. (c) Conjugation of LPETG-tagged probe or protein to naturally exposed N-terminal glycine residues at the surface of cells. (d–g) Saccharomyces cerevisiae , Toxoplasma gondii , human embryonic kidney HEK 293 T cells, or total mouse splenocytes from C57BL/6 mice were incubated for 1 h at room temperature (RT) with or without 500 μM of biotin-LPETG and with or without 20 μM of sortase A. Conjugation of the biotin-LPETG probe was analyzed by SDS PAGE followed by immunoblotting using streptavidin-HRP. *Endogenous biotinylated proteins. (h) Total splenocytes from C57BL/6 mice were incubated for 1 h at RT with 500 μM of biotin-LPETG in the presence (dark gray histograms) or absence (light gray histograms) of 20 μM of sortase A. Conjugation of biotin-LPETG was analyzed by flow cytometry using streptavidin–phycoerythrin (PE) together with antibodies specific for T cells (TCRβ), B cells (CD19), or erythrocytes (Ter119). (i) Erythrocyte-depleted splenocytes were incubated with 20 μM sortase A and 500 μM biotin-LPETG for the indicated times. Cells were washed and incubated with streptavidin-PE and analyzed by flow cytometry. Scatter plots show the mean fluorescence intensity of streptavidin-PE staining for each time point, normalized to maximum staining (120 min).
Figure Legend Snippet: Sortagging of genetically unmanipulated cells. (a) Conjugation of G (n) -probe to the C-terminus of an LPETG-tagged protein using sortase A. (b) Conjugation of probe-LPETG to the N-terminus of a G (n) -tagged protein using sortase A. (c) Conjugation of LPETG-tagged probe or protein to naturally exposed N-terminal glycine residues at the surface of cells. (d–g) Saccharomyces cerevisiae , Toxoplasma gondii , human embryonic kidney HEK 293 T cells, or total mouse splenocytes from C57BL/6 mice were incubated for 1 h at room temperature (RT) with or without 500 μM of biotin-LPETG and with or without 20 μM of sortase A. Conjugation of the biotin-LPETG probe was analyzed by SDS PAGE followed by immunoblotting using streptavidin-HRP. *Endogenous biotinylated proteins. (h) Total splenocytes from C57BL/6 mice were incubated for 1 h at RT with 500 μM of biotin-LPETG in the presence (dark gray histograms) or absence (light gray histograms) of 20 μM of sortase A. Conjugation of biotin-LPETG was analyzed by flow cytometry using streptavidin–phycoerythrin (PE) together with antibodies specific for T cells (TCRβ), B cells (CD19), or erythrocytes (Ter119). (i) Erythrocyte-depleted splenocytes were incubated with 20 μM sortase A and 500 μM biotin-LPETG for the indicated times. Cells were washed and incubated with streptavidin-PE and analyzed by flow cytometry. Scatter plots show the mean fluorescence intensity of streptavidin-PE staining for each time point, normalized to maximum staining (120 min).

Techniques Used: Conjugation Assay, Mouse Assay, Incubation, SDS Page, Flow Cytometry, Cytometry, Fluorescence, Staining

Installation of VHHs on mouse lymphocytes. (a) In vitro activated CD8 T cells from OTI RAG–/– mice were incubated for 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETG or VHH7-LPETG and with or without 20 μM of sortase A. Control or sortagged cells were incubated with purified GFP. Binding of GFP was analyzed by flow cytometry. (b) Control or sortagged cells were incubated with purified GFP. The amount of bound GFP was estimated by analysis of cell lysates by SDS-PAGE and immunoblotting against GFP and comparing the resultant signal to a GFP standard (right lanes). (c) Erythrocyte-depleted splenocytes were incubated with or without 500 μM enhancer-LPETG and 20 μM sortase A. After 60 min, 500 μM biotin-LPETG was added to reactions where indicated for a further 15 min. Dot plots show the binding of APC-conjugated streptavidin and GFP by sortagged cells after washing. (d) In vitro activated CD8 T cells from OTI RAG–/– mice were incubated for 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETG or VHH7-LPETG and 20 μM of sortase A. Sortagged cells were incubated with splenocytes from WT mice for 20 h. Histograms show the percentage of propidium iodide-negative CD4 and CD19 cells, compared to cells incubated with control activated OTI CD8 T cells. Error bars: standard deviation ( n = 3). (e) CAR T cells are genetically engineered to express a synthetic receptor composed of an extracellular single-chain variable fragment and one or several cytoplasmic activating motifs that mediate signal transduction and T cell activation upon antigen binding. (f) Sortase-mediated conjugation of VHHs on activated T cells affords redirection of cytotoxicity toward cells expressing the targeted antigen.
Figure Legend Snippet: Installation of VHHs on mouse lymphocytes. (a) In vitro activated CD8 T cells from OTI RAG–/– mice were incubated for 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETG or VHH7-LPETG and with or without 20 μM of sortase A. Control or sortagged cells were incubated with purified GFP. Binding of GFP was analyzed by flow cytometry. (b) Control or sortagged cells were incubated with purified GFP. The amount of bound GFP was estimated by analysis of cell lysates by SDS-PAGE and immunoblotting against GFP and comparing the resultant signal to a GFP standard (right lanes). (c) Erythrocyte-depleted splenocytes were incubated with or without 500 μM enhancer-LPETG and 20 μM sortase A. After 60 min, 500 μM biotin-LPETG was added to reactions where indicated for a further 15 min. Dot plots show the binding of APC-conjugated streptavidin and GFP by sortagged cells after washing. (d) In vitro activated CD8 T cells from OTI RAG–/– mice were incubated for 1 h at RT with or without 500, 50, or 5 μM of enhancer-LPETG or VHH7-LPETG and 20 μM of sortase A. Sortagged cells were incubated with splenocytes from WT mice for 20 h. Histograms show the percentage of propidium iodide-negative CD4 and CD19 cells, compared to cells incubated with control activated OTI CD8 T cells. Error bars: standard deviation ( n = 3). (e) CAR T cells are genetically engineered to express a synthetic receptor composed of an extracellular single-chain variable fragment and one or several cytoplasmic activating motifs that mediate signal transduction and T cell activation upon antigen binding. (f) Sortase-mediated conjugation of VHHs on activated T cells affords redirection of cytotoxicity toward cells expressing the targeted antigen.

Techniques Used: In Vitro, Mouse Assay, Incubation, Purification, Binding Assay, Flow Cytometry, Cytometry, SDS Page, Standard Deviation, Transduction, Activation Assay, Conjugation Assay, Expressing

33) Product Images from "The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis"

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00222

FK506-binding protein 13 (FKBP13) promotes ubiquitination-mediated degradation of immunoglobin (Ig) molecules . (A) J558 cells were lysed, immunoprecipitated with anti-goat IgG, anti-FKBP13, or anti-IgA antibodies, and assayed by western blotting. (B–G) J558 cells were transfected with MigR1, MigR1-myc-FKBP13 (FKBP13), pGFP-V-RS-shFKBP13 (shFKBP13), or pGFP-V-RS-SCR (SCR) as indicated. (B) The transfectants were incubated with 1 μM MG-132 or DMSO for 24 h followed by western blotting. (C) Lysates from the transfectants were immnoprecipitated with anti-IgA antibodies and assayed by western blotting. (D,E) GFP + transfectants were sorted by FACS and cultured for 24 h, and supernatant IgA was assayed by enzyme-linked immunosorbent assays. (F) IgA Cα transcripts in the transfectants were assayed by quantitative RT-PCR. (G) J558 cells were transfected with MigR1 or MigR1-myc-FKBP13 constructs together with reporter constructs (pGL3b-UPRE and pRL-CMV), and dual luciferase activities were measured. Firefly luciferase activity was normalized by the Renilla luciferase activity and is shown as relative luciferase units (RLU). All data are representative of at least three independent experiments. * p
Figure Legend Snippet: FK506-binding protein 13 (FKBP13) promotes ubiquitination-mediated degradation of immunoglobin (Ig) molecules . (A) J558 cells were lysed, immunoprecipitated with anti-goat IgG, anti-FKBP13, or anti-IgA antibodies, and assayed by western blotting. (B–G) J558 cells were transfected with MigR1, MigR1-myc-FKBP13 (FKBP13), pGFP-V-RS-shFKBP13 (shFKBP13), or pGFP-V-RS-SCR (SCR) as indicated. (B) The transfectants were incubated with 1 μM MG-132 or DMSO for 24 h followed by western blotting. (C) Lysates from the transfectants were immnoprecipitated with anti-IgA antibodies and assayed by western blotting. (D,E) GFP + transfectants were sorted by FACS and cultured for 24 h, and supernatant IgA was assayed by enzyme-linked immunosorbent assays. (F) IgA Cα transcripts in the transfectants were assayed by quantitative RT-PCR. (G) J558 cells were transfected with MigR1 or MigR1-myc-FKBP13 constructs together with reporter constructs (pGL3b-UPRE and pRL-CMV), and dual luciferase activities were measured. Firefly luciferase activity was normalized by the Renilla luciferase activity and is shown as relative luciferase units (RLU). All data are representative of at least three independent experiments. * p

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, FACS, Cell Culture, Quantitative RT-PCR, Construct, Luciferase, Activity Assay

34) Product Images from "Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients"

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

Journal: Oncotarget

doi: 10.18632/oncotarget.11181

Immunofluorescence reactivity of IgG from patient sera on LoVo cell line ADAM10 pro-domain: reactivity of the rabbit polyclonal anti-ADAM10 pro-domain-specific Ab (ab39178, Abcam), which recognized the immature non-functional isoform of ADAM10, showed patched reactivity. ADAM10 ectodomain: reactivity of the goat polyclonal anti-ADAM10 ectodomain-specific Ab (R D-Systems, AB936), which recognized both the immature and mature (cleaved and functional) isoforms of ADAM10, showed patched and diffuse signals. Anti-ADAM10 auto-Ab positive serum: reactivity of the human IgG fraction purified from a representative serum (C2) of Crc patients considered positive for the presence of auto-Ab anti ADAM10, showed patched reactivity. Anti-ADAM10 auto-Ab negative serum: no reactivity was observed for the human IgG fraction purified from serum (C25) of a representative Crc patient considered negative for the presence of auto-Ab anti ADAM10. Cell nuclei were stained with Hoechst-33342. The reactivity of the secondary antibodies (goat anti-rabbit IgG Alexa-488; donkey anti-goat IgG Alexa-488 and goat anti-human IgG FITC) are shown as negative controls in SM-Figure 3 . Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.
Figure Legend Snippet: Immunofluorescence reactivity of IgG from patient sera on LoVo cell line ADAM10 pro-domain: reactivity of the rabbit polyclonal anti-ADAM10 pro-domain-specific Ab (ab39178, Abcam), which recognized the immature non-functional isoform of ADAM10, showed patched reactivity. ADAM10 ectodomain: reactivity of the goat polyclonal anti-ADAM10 ectodomain-specific Ab (R D-Systems, AB936), which recognized both the immature and mature (cleaved and functional) isoforms of ADAM10, showed patched and diffuse signals. Anti-ADAM10 auto-Ab positive serum: reactivity of the human IgG fraction purified from a representative serum (C2) of Crc patients considered positive for the presence of auto-Ab anti ADAM10, showed patched reactivity. Anti-ADAM10 auto-Ab negative serum: no reactivity was observed for the human IgG fraction purified from serum (C25) of a representative Crc patient considered negative for the presence of auto-Ab anti ADAM10. Cell nuclei were stained with Hoechst-33342. The reactivity of the secondary antibodies (goat anti-rabbit IgG Alexa-488; donkey anti-goat IgG Alexa-488 and goat anti-human IgG FITC) are shown as negative controls in SM-Figure 3 . Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.

Techniques Used: Immunofluorescence, Functional Assay, Purification, Staining, Microscopy, Software

Immunofluorescence reactivity after competition with anti ADAM10 pro-domain Ab Anti-ADAM10 pro-domain: reactivity of the rabbit anti-ADAM10 pro-domain Ab (ab39178, Abcam) after competition with control rabbit IgG. Anti-ADAM10 positive serum (C2): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered positive for the presence of auto-Abs anti ADAM10 after competition either with control rabbit IgGs or the anti-ADAM10 pro-domain Ab. Anti-ADAM10 negative serum (C32): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered negative for the presence of anti ADAM10 auto-Ab after competition either with control rabbit IgGs or anti-ADAM10 pro-domain Ab. Cell nuclei were stained with Hoechst-33342; secondary Abs were goat anti-rabbit IgG Alexa-488 and goat anti-human IgG FITC. Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.
Figure Legend Snippet: Immunofluorescence reactivity after competition with anti ADAM10 pro-domain Ab Anti-ADAM10 pro-domain: reactivity of the rabbit anti-ADAM10 pro-domain Ab (ab39178, Abcam) after competition with control rabbit IgG. Anti-ADAM10 positive serum (C2): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered positive for the presence of auto-Abs anti ADAM10 after competition either with control rabbit IgGs or the anti-ADAM10 pro-domain Ab. Anti-ADAM10 negative serum (C32): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered negative for the presence of anti ADAM10 auto-Ab after competition either with control rabbit IgGs or anti-ADAM10 pro-domain Ab. Cell nuclei were stained with Hoechst-33342; secondary Abs were goat anti-rabbit IgG Alexa-488 and goat anti-human IgG FITC. Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.

Techniques Used: Immunofluorescence, Purification, Staining, Microscopy, Software

Co-localization of immunofluorescence signals from anti-ADAM10 pro-domain Ab and IgG from anti-ADAM10 positive patient serum Double staining was performed with rabbit anti-ADAM10 pro-domain (ab39178, Abcam), and either IgGs purified from Crc patient serum or mouse anti-HLA class I (Santa-Cruz Biotechnology). Secondary Abs were Alexa-546-conjugated goat-anti-rabbit IgG and Alexa-488-conjugated rabbit-anti-mouse IgG or FITC-conjugated goat-anti-human IgG. Cell nuclei were stained with Hoechst-33342, and differential interference contrast (DIC) images were also acquired. Staining was assessed by immunofluorescence confocal microscopy (Leica TCS SP5 Laser Scanning Confocal) and images acquired with LAS-AF (Leica) software; magnification 63X. Images from single channel and merged images are shown. Images were linearly adjusted for brightness and contrast using ImageJ 1.47v software.
Figure Legend Snippet: Co-localization of immunofluorescence signals from anti-ADAM10 pro-domain Ab and IgG from anti-ADAM10 positive patient serum Double staining was performed with rabbit anti-ADAM10 pro-domain (ab39178, Abcam), and either IgGs purified from Crc patient serum or mouse anti-HLA class I (Santa-Cruz Biotechnology). Secondary Abs were Alexa-546-conjugated goat-anti-rabbit IgG and Alexa-488-conjugated rabbit-anti-mouse IgG or FITC-conjugated goat-anti-human IgG. Cell nuclei were stained with Hoechst-33342, and differential interference contrast (DIC) images were also acquired. Staining was assessed by immunofluorescence confocal microscopy (Leica TCS SP5 Laser Scanning Confocal) and images acquired with LAS-AF (Leica) software; magnification 63X. Images from single channel and merged images are shown. Images were linearly adjusted for brightness and contrast using ImageJ 1.47v software.

Techniques Used: Immunofluorescence, Double Staining, Purification, Staining, Confocal Microscopy, Software

35) Product Images from "Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells"

Article Title: Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells

Journal: Oncotarget

doi:

NTRK1-positive neuroblastoma cells attract Schwann cells and induce their proliferation by secreting NRG1 (A) Dual immunofluorescence staining using anti-rat NGFR primary antibody followed by goat anti-mouse IgG-FITC secondary antibody and DAPI counterstaining in Schwann cells in short-term culture from P3 rats (scale bar 50μm). (B) Bars represent the fractions of NGFR-positive and -negative cells from the total population of Schwann cells before and after positive selection using anti-rat NGFR primary antibody and rat anti-mouse IgG1 microbeads. (C) Growth curves of Schwann cells over a period of 80h as detected by electric cell-substrate impedance sensing in real-time following addition of medium conditioned by SY5Y-NTRK1 cells (NTRK1 CM), recombinant NRG1 + forskolin (NRG1 + forskolin), recombinant NRG1 (NRG1) or medium without additives (no additives). (D) Growth curves of Schwann cells over a period of 80h as detected by electric cell-substrate impedance sensing in real-time following addition of medium conditioned by SY5Y-NTRK1 cells (NTRK1 CM), medium conditioned by SY5Y-NTRK1 cells supplemented with anti-NRG1 isotype control (NTRK1 CM + isotype ctrl) or anti-NRG1 antibody (NTRK1 CM + anti-NRG1) or medium conditioned by SY5Y-vec cells (vec CM). For all growth curves in (C) and (D), cell index was normalized each at a base-time of 24h following plating of Schwann cells using an RTCA software-based algorithm. *p
Figure Legend Snippet: NTRK1-positive neuroblastoma cells attract Schwann cells and induce their proliferation by secreting NRG1 (A) Dual immunofluorescence staining using anti-rat NGFR primary antibody followed by goat anti-mouse IgG-FITC secondary antibody and DAPI counterstaining in Schwann cells in short-term culture from P3 rats (scale bar 50μm). (B) Bars represent the fractions of NGFR-positive and -negative cells from the total population of Schwann cells before and after positive selection using anti-rat NGFR primary antibody and rat anti-mouse IgG1 microbeads. (C) Growth curves of Schwann cells over a period of 80h as detected by electric cell-substrate impedance sensing in real-time following addition of medium conditioned by SY5Y-NTRK1 cells (NTRK1 CM), recombinant NRG1 + forskolin (NRG1 + forskolin), recombinant NRG1 (NRG1) or medium without additives (no additives). (D) Growth curves of Schwann cells over a period of 80h as detected by electric cell-substrate impedance sensing in real-time following addition of medium conditioned by SY5Y-NTRK1 cells (NTRK1 CM), medium conditioned by SY5Y-NTRK1 cells supplemented with anti-NRG1 isotype control (NTRK1 CM + isotype ctrl) or anti-NRG1 antibody (NTRK1 CM + anti-NRG1) or medium conditioned by SY5Y-vec cells (vec CM). For all growth curves in (C) and (D), cell index was normalized each at a base-time of 24h following plating of Schwann cells using an RTCA software-based algorithm. *p

Techniques Used: Immunofluorescence, Staining, Selection, Electric Cell-substrate Impedance Sensing, Recombinant, Software

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Flow Cytometry:

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Article Snippet: .. The following specific rat anti-mouse monoclonal antibodies were used for flow cytometry: PE-conjugated CD11b (clone 3A33, SouthernBiotech, Birmingham, AL: 1 μg/mL), FITC-conjugated CD3 (T-lymphocytes, clone:145-2C11, BioLegend, San Diego, CA: 5 μg/mL), Brilliant Violet 421-conjugated NK-1.1 (natural killer cells, clone: PK136, BioLegend: 5 μg/mL), APC-conjugated CD115 (clone:AFS98, monocytes: CD115+; neutrophils: CD115-, eBioscience, San Diego, CA: 2 μg/mL), and PerCP-Cy5.5-conjugated CD19 (B-lymphocytes, clone:1D3, BD Biosciences, San Jose, CA: 2 μg/mL). .. Cell sorting was performed using an LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Cytometry:

Article Title: The pathogenic relevance of αM-integrin in Guillain-Barré syndrome
Article Snippet: .. The following specific rat anti-mouse monoclonal antibodies were used for flow cytometry: PE-conjugated CD11b (clone 3A33, SouthernBiotech, Birmingham, AL: 1 μg/mL), FITC-conjugated CD3 (T-lymphocytes, clone:145-2C11, BioLegend, San Diego, CA: 5 μg/mL), Brilliant Violet 421-conjugated NK-1.1 (natural killer cells, clone: PK136, BioLegend: 5 μg/mL), APC-conjugated CD115 (clone:AFS98, monocytes: CD115+; neutrophils: CD115-, eBioscience, San Diego, CA: 2 μg/mL), and PerCP-Cy5.5-conjugated CD19 (B-lymphocytes, clone:1D3, BD Biosciences, San Jose, CA: 2 μg/mL). .. Cell sorting was performed using an LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Blocking Assay:

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Article Snippet: .. After blocking with a 50 μl blocking buffer [0.5% bovine serum albumin (BSA), 0.05% Tween-20, and 1 mM EDTA in PBS], 50 μl of twofold serially diluted samples were added and incubated at room temperature for 2 h. Following three washes, horseradish peroxidase-labeled goat anti-mouse IgG or rat anti-mouse IgA (Southern Biotech) was added, and the plate was incubated at room temperature for 2 h. The color was developed using tetramethylbenzidine (Science Products, Inc.). ..

SYBR Green Assay:

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Incubation:

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other:

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Staining:

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