Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of PAI ‐1 with PAI ‐1 si RNA /sh RNA reduces p53 and p21 protein levels, increases Rb phosphorylation, and attenuates bleomycin‐induced L2 cell senescence. Rat ATII (L2) cells were treated with 50 mU /mL bleomycin for 24 h (A & B) and then cultured in bleomycin‐free medium for additional 72 h (B). (C) L2 cell was transfected with PAI ‐1 si RNA or nontarget si RNA ( NT si RNA ). D‐N) PAI ‐1 sh RNA or NT sh RNA stably transfected L2 cells were treated with bleomycin for 24 h and then cultured in bleomycin‐free medium for additional 24 (D–L) or 72 (M and N) hours. PAI ‐1, serine‐18 phosphorylated p53 (p53 S−18P ), p53, p21, and phosphorylated Rb (ppRb) proteins were determined by Westerns. β‐Actin is used as loading control. D, representative Western blotting pictures; E‐J, semi‐quantified band intensities normalized by β‐actin. (K and L) Immunostaining and quantification of proliferating cell nuclear antigen ( PCNA ). (M and N) SA ‐β‐gal activity revealed by X‐gal staining. α, Significantly different from corresponding saline‐treated cells; β, significantly different from bleomycin‐treated NT sh RNA ‐transfected cells; ζ, significantly different from corresponding NT sh RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Cell Culture, Transfection, Stable Transfection, Western Blot, Immunostaining, Activity Assay, Staining

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Inhibition of PAI ‐1 activity with a small molecule PAI ‐1 inhibitor TM 5275 attenuates bleomycin‐induced L2 cell senescence. L2 cells were treated with 50 mU /mL bleomycin in the presence or absence of 25 μ m of TM 5275 for 24 hours and then cultured in bleomycin‐free medium for additional 72 (A and B) or 24 (C–G) hours. (A and B) SA ‐β‐gal activity was revealed by X‐gal staining; (C–G) Western analyses of the proteins of interested in cell lysates, the band intensities semi‐quantified by ImageJ software, and normalized by β‐actin. α, Significantly different from corresponding saline‐treated cells; β, significantly different from bleomycin‐treated vehicle controls ( P < 0.05, n = 3).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Inhibition, Activity Assay, Cell Culture, Staining, Western Blot, Software

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of PAI ‐1 protein with PAI ‐1 sh RNA attenuates doxorubicin‐induced L2 cell senescence. PAI ‐1 sh RNA or NT sh RNA stably transfected L2 cells were treated with 50 n m of doxorubicin (Dox)/saline for 24 h and cultured in doxorubicin‐free medium for additional 24 h (A–D) or 72 h (E and F). PAI ‐1, p53, p21, and β‐actin in cell lysates were determined by Westerns. A, representative Western blotting pictures; B–D, semi‐quantified band intensities by ImageJ program and normalized by β‐actin. E and F, SA ‐β‐gal activity was revealed by X‐gal staining. α, Significantly different from the corresponding saline‐treated cells; β, significantly different from doxorubicin‐treated NT sh RNA ‐transfected cells; ζ, significantly different from saline‐treated NT sh RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Stable Transfection, Transfection, Cell Culture, Western Blot, Activity Assay, Staining

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of p53 protein with p53 si RNA abrogates PAI ‐1 protein‐mediated L2 cell senescence. (A and B) L2 cells were treated with 1 μg/mL of hPAI ‐1, dissolved in 0.1% BAS , or 0.1% bovine serum albumin ( BSA ) for 72 h. (A) PAI ‐1 mRNA was determined by real‐time PCR ; (B) Proteins of interest were determined by Westerns. (C–I) L2 cells were transfected with p53 si RNA or nontarget si RNA ( NT si RNA ) and then treated with hPAI ‐1 or BSA for 72 h. (C and D) SA ‐β‐gal activity was measured by X‐gal staining. (E–I) Western analyses of proteins of interest; E, representative Western blotting pictures; F–I, semi‐quantified band intensities normalized by β‐actin. α, Significantly different from corresponding 0.1% BSA (solvent) controls; β, significantly different from hPAI ‐1‐treated NT si RNA ‐transfected cells; ζ, significantly different from BSA ‐treated NT si RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Activity Assay, Staining, Western Blot

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockout of the PAI ‐1 gene specifically in ATII cells in mice attenuates bleomycin‐induced ATII cell senescence in vivo . (A and B) Double immunostaining of isolated ATII cells with anti‐ PAI ‐1 and anti‐p53 antibodies. (C and D) Double immunostaining of isolated ATII cells with anti‐p21 and anti‐ SPC antibodies. (E and F) SA ‐β‐gal activity in freshly isolated mouse ATII cells was revealed by X‐gal staining. Left panels are representative SA ‐β‐gal staining pictures; right panel is quantitative data. (G–M) Western analyses of the proteins of interest in isolated ATII cells. (N–S) Double‐immunofluorescence staining of mouse lung tissues with PAI ‐1, p53, or p21 and ATII cell marker SPC . Top panels are representative Western blotting pictures, and bottom panels are quantitative data. α, Significantly different from same genotype, saline‐treated mice; β, significantly different from bleomycin‐treated PAI ‐1 fl/fl mice; ζ, significantly different from saline‐treated PAI ‐1 fl/fl mice ( P < 0.05, n = 3–6).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Knock-Out, In Vivo, Double Immunostaining, Isolation, Activity Assay, Staining, Western Blot, Double Immunofluorescence Staining, Marker

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Deletion of the PAI ‐1 gene specifically in ATII cells in mice attenuates bleomycin‐induced lung fibrosis. (A) Body weight changes before and 14 days after bleomycin/saline treatment. (B) The amount of PAI ‐1 protein in BAL fluid measured by ELISA . (C) Trichrome staining of collagen and (D) Sirius red staining of collagen. (E) Hydroxyproline content in mouse lung measured using the Hydroxyproline Assay Kit (Chrondrex, Inc) and expressed as % of hydroxyproline in saline‐treated fl/fl mice. (F–I) Western analyses of procollagen 1α1, procollagen 1α2, and alpha‐smooth muscle actin (α‐ SMA ) in mouse lung tissue. α, Significantly different from same genotype, saline‐treated mice; β, significantly different from bleomycin‐treated PAI ‐1 fl/fl mice ( P < 0.05, n = 3–8).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Hydroxyproline Assay, Western Blot

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Tissue plasminogen activator (t-PA) is present on the outer leaflet of the plasma membrane of stimulated chromaffin cells. ( a ) Dual labeling of cell surface t-PA and exocytotic sites. Cells were stimulated with nicotine 20 µM in the presence of anti-t-PA and anti-DBH antibodies. Cells were then fixed and incubated with secondary antibodies coupled to Alexa Fluor ® 488 and Alexa Fluor ® 551, respectively. Confocal images were recorded in the same optical section and with the same parameters of lasers and photomultipliers. Scale bar: 10 µM. ( b ) The t-PA labeling on the surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine without (S) or after EGTA wash (S + EGTA). Anti-t-PA antibodies were revealed with Alexa Fluor ® 488-conjugated anti-rabbit antibodies and F-actin with TRITC-phalloidin to visualize the cell shape. Confocal images were recorded in the same optical section by a dual exposure procedure. Scale bar: 10 µM. ( c ) Semi-quantitative analysis of t-PA labeling on the cell surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine (S) without (Control) or after EGTA wash (S + EGTA). The t-PA labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (*** = p < 0.001, ** = p < 0.01). Three experiments were done on independent cell cultures and pooled ( n = 36 and 65 control cells, 40 and 75 EGTA-treated cells for resting and stimulated conditions, respectively). ( d ) Lysate, secreted material and EGTA eluate from chromaffin cells in the resting condition (R) or stimulated with nicotine 20 µM (S) were analyzed by western blot and revealed with anti-t-PA and anti-AnxA2 antibodies. Data correspond to a typical experiment representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Incubation, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Membrane topography of AnxA2, t-PA and exocytotic sites after immunogold labeling of the outer face of the plasma membrane sheets prepared from stimulated chromaffin cells. ( a ) Plasma membrane sheets were prepared from bovine chromaffin cells stimulated by nicotine for 5 min. To label DBH, AnxA2 and t-PA exposed at the surface of cells undergoing exocytosis, anti-DBH, anti-AnxA2 and anti-t-PA antibodies were added during stimulation. Membrane sheets were labeled with anti-mouse antibodies coupled to 10 nm gold particles to detect DBH antibodies revealing exocytotic sites (red circle) and rabbit antibodies coupled to 15 nm gold particles to label AnxA2 or t-PA (green circle). ( b ) The histogram represents the relative distribution of 15 nm gold particles as a function of their distance from the granule membrane once inserted in the plasma membrane (blue line). The distance was measured and the number of particles was counted manually with Photoshop. Three experiments were done on independent cell cultures ( c ). Double staining experiment for t-PA (10 nm gold particles) and AnxA2 (15 nm gold particles) were performed with the same protocol. Scale bar: 100 nm. ( d ) The histogram represents the relative distribution of 10 nm gold particles (t-PA) as a function of their distance from 15 nm gold particles (AnxA2). The distance was measured and the number of particles was counted manually with Photoshop. Two experiments were done on two independent cell cultures.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Double Staining

Journal:

Article Title: Remodeling of the Vessel Wall after Copper-Induced Injury Is Highly Attenuated in Mice with a Total Deficiency of Plasminogen Activator Inhibitor-1

doi:

Figure Lengend Snippet: Transmission electron microscopy of carotid arteries of WT and PAI-1−/− mice 3 days after cuff implantation. A: A neutrophil with vesicular transport activity (arrow) is seen attached to the luminal endothelium in WT carotid artery close to a cellular junction (asterisk) (original magnification, ×11,500). B: A polymorphonuclear leukocyte (arrow) is present in an open subendothelial space in WT carotid artery (original magnification, ×6,200). A and B suggest that a diapedesis process is occurring. C: Altered cellular morphology of an endothelial cell (E) is evident in PAI-1−/− carotid artery, the result of copper-induced injury (original magnification, ×6,800). D: Activated macrophages with visible phagolysosomes (P) are evident in the medial compartment of a PAI-1−/− carotid artery (original magnification, ×2,700).

Article Snippet: PAI-1 expression in the vessel wall was evaluated by immunohistochemistry using a rabbit anti-murine PAI-1 polyclonal antibody (Molecular Innovations, Inc., Royal Oak, MI).

Techniques: Transmission Assay, Electron Microscopy, Activity Assay

Journal:

Article Title: Remodeling of the Vessel Wall after Copper-Induced Injury Is Highly Attenuated in Mice with a Total Deficiency of Plasminogen Activator Inhibitor-1

doi:

Figure Lengend Snippet: Histological analysis of carotid arteries from WT and PAI-1−/− mice 7 days after implantation of the copper cuff. A: H&E stain of WT carotid artery demonstrates a reactive adventitial (asterisk) and medial (arrow) compartments with the presence of a small neointima (arrowhead). Original magnification, ×200. Similar H&E staining of a carotid artery from a PAI-1−/− mouse (B) shows a quiescent state of these vascular compartments. Original magnification, ×200. C: Anti-α-actin immunostain of a WT carotid artery demonstrates enhanced smooth muscle cells in the medial compartment (arrow) (original magnification, ×200), whereas a PAI-1−/− carotid artery (D) indicates no apparent change in the smooth muscle cell population in the medial compartment. Original magnification, ×200. E: CD45 immunostain of WT carotid artery demonstrates a leukocyte-enriched adventitia (arrow). Original magnification, ×200. F: CD45 immunostain of WT carotid artery demonstrates leukocytes within the neointima (arrow). Original magnification, ×400. G: Anti-fibrin(ogen) immunostain of WT carotid artery shows fibrin-rich medial (arrow) and adventitial (asterisk) compartments (original magnification, ×200), whereas similar immunostaining of a PAI-1−/− carotid artery (H) demonstrates no apparent fibrin deposition in the vessel wall. Original magnification, ×200.

Article Snippet: PAI-1 expression in the vessel wall was evaluated by immunohistochemistry using a rabbit anti-murine PAI-1 polyclonal antibody (Molecular Innovations, Inc., Royal Oak, MI).

Techniques: Staining, Immunostaining

Journal:

Article Title: Remodeling of the Vessel Wall after Copper-Induced Injury Is Highly Attenuated in Mice with a Total Deficiency of Plasminogen Activator Inhibitor-1

doi:

Figure Lengend Snippet: Vessel counts per mm 2 area of the adventitial compartment of arteries from WT and PAI-1−/− mice 7 and 21 days after perivascular placement of the cuff. WT versus PAI-1−/− at day 21, P = 0.0057.

Article Snippet: PAI-1 expression in the vessel wall was evaluated by immunohistochemistry using a rabbit anti-murine PAI-1 polyclonal antibody (Molecular Innovations, Inc., Royal Oak, MI).

Techniques:

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator mediates neuronal detection and adaptation to metabolic stress

doi: 10.1038/jcbfm.2013.124

Figure Lengend Snippet: Glucose deprivation (GD) induces the release of tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons. (A) Representative micrograph of wild-type (Wt) cerebral cortical neurons maintained during 5 minutes under 25 mmol/L (+G) or 0 mmol/L (−G) of glucose and stained with antibodies against the postsynaptic dendritic marker anti-microtubule associated protein (MAP-2) (blue) and tPA (red). Green corresponds to DNA (Hoechst). Magnification × 20 in (a, e) and × 60 in (b–d) and (f–h). (B) Representative micrograph of Wt cerebral cortical neurons maintained during 5 minutes under GD and stained with antibodies against MAP-2 (blue), tPA (red), and synaptophysin (green). Arrows denote the presence of tPA-containing synaptophysin-positive presynaptic vesicles in direct contact with the postsynaptic, MAP-2-positive, dendrite. Magnification × 60. (C) Mean concentration of tPA in the culture medium of Wt cerebral cortical neurons exposed during 0 to 5 minutes to either oxygen deprivation (OD, black circles), or GD (black triangles), or oxygen and glucose deprivation (OGD; black squares), or kept under normal glucose and oxygen concentrations after medium change (baseline; white diamonds). Lines denote s.d. n=10 per condition; *P<0.05 compared with GD and OD; ^P<0.05 compared with OD and OGD; §P<0.05 compared with sister cultures maintained under physiologic conditions and with cultures exposed to either GD or OGD conditions.

Article Snippet: Recombinant murine tPA, proteolytically inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A), human Lys plasmin, an ELISA kit that detects active tPA, and sheep anti-tPA antibodies (Cat # SASMTPA) were acquired from Molecular Innovations (Novi, MI, USA).

Techniques: Staining, Marker, Concentration Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator mediates neuronal detection and adaptation to metabolic stress

doi: 10.1038/jcbfm.2013.124

Figure Lengend Snippet: Tissue-type plasminogen activator (tPA) induces adenosine monophosphate-activated protein kinase (AMPK) activation in the postsynaptic terminal via N-methyl-D-aspartate receptors (NMDARs) activation. (A) Representative western blot analysis of pAMPK expression in non-ischemic wild-type (Wt) cerebral cortical synaptoneurosomes after 5 minutes of incubation with 5 nmol/L of tPA (+) or vehicle (control; −). (B) Representative microphotograph of Wt cerebral cortical neurons incubated during 5 minutes with vehicle (control; a, c, e, and g) or 5 nmol/L of tPA (b, d, f, and h), and stained with antibodies against the dendritic marker anti-microtubule associated protein (MAP-2) (red) and pAMPK (green). Magnification × 40 in (a, b) and × 60 in (c–h). (C) Representative microphotograph of Wt cerebral cortical neurons incubated 5 minutes with 5 nmol/L of tPA and stained with antibodies against synaptophysin (red) and pAMPK (green). Arrows denote examples where presynaptic synaptophysin-positive vesicles are in juxtaposition with postsynaptic pAMPK. Magnification × 100. (D) Representative western blot analysis of pAMPK expression in cerebral cortical synaptoneurosomes prepared from the cerebral cortex of Wt mice after transient middle cerebral artery occlusion (tMCAO) and treatment with either 0.9 mg/kg/IV of recombinant tPA (rtPA) or a comparable volume of saline solution. (E) Representative western blot analysis of the expression NR2A and NR2B subunits of NMDARs in synaptoneurosomes prepared from the cerebral cortex of Wt mice. (F) Representative western blot analysis of pAMPK expression in cerebral cortical synaptoneurosomes prepared from the cerebral cortex of Wt mice 5 minutes after tMCAO and treatment with 0.9 mg/kg/IV of rtPA alone or in combination with 2 μg of MK-801.

Article Snippet: Recombinant murine tPA, proteolytically inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A), human Lys plasmin, an ELISA kit that detects active tPA, and sheep anti-tPA antibodies (Cat # SASMTPA) were acquired from Molecular Innovations (Novi, MI, USA).

Techniques: Activation Assay, Western Blot, Expressing, Incubation, Staining, Marker, Recombinant

Journal: The Journal of Neuroscience

Article Title: Localization and Regulation of the Tissue Plasminogen Activator–Plasmin System in the Hippocampus

doi: 10.1523/JNEUROSCI.22-06-02125.2002

Figure Lengend Snippet: tPA is abundantly expressed in the mossy fiber pathway. A, B, tPA immunohistochemistry on wild-type (wt) sections shows robust tPA staining (red) in the hippocampal mossy fiber pathway. Sections are counter-stained with hematoxylin to visualize the neuronal cell body layers of the hippocampus. DG, Dentate gyrus;MF, mossy fiber pathway; n = 20 animals. C, D, tPA immunostain without the hematoxylin counterstain showing lack of significant staining within the neuronal cell bodies (compare high-magnification B withD). Arrows in D highlight vascular endothelial cell staining that is seen throughout the brain.E, F, tPA immunohistochemistry on wild-type sections after preabsorbing the antibody (Figure legend continues.) (Figure legend continued.) with tPA as described in Materials and Methods. Notice the loss of mossy fiber and vascular staining but residual background parenchymal stain;n = 7 animals. G, tPA immunohistochemistry on tPA-deficient (tPA−/−) brain sections. Notice a similar level of background parenchymal staining compared with E; n = 10 animals.H, Zymographic analysis of tPA activity (dark zone of lysis against a milky background) closely parallels staining seen by immunohistochemistry;n = 20 animals. Activity is plasminogen-dependent (data not shown). Boxes in A,C, and E show the region magnified inB, D, and F.Arrowheads in E–G highlight background CA2 cell body staining.

Article Snippet: Antibodies used were as follows: rabbit anti-human tPA antisera ( Waller and Schleuning, 1985 ; or Molecular Innovations, catalog #ASHTPA), sheep anti-mouse PAI-1 (American Diagnostica, catalog #1040), sheep anti-rat plasminogen antisera (a kind gift from E. Reich, State University of New York, Stony Brook, NY), and neuroserpin antisera (a gift from D. Lawrence, American Red Cross, Rockville, MD).

Techniques: Immunohistochemistry, Staining, Activity Assay, Lysis

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Endogenous expression of C3 is very low in human B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, PBMCs, and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.

Article Snippet: Antibodies (Abs) used to study C3 expression were the followings: polyclonal goat anti-human C3 (Quidel, #A304) and polyclonal goat anti-human C3 (Calbiochem, #204869).

Techniques: Expressing, Isolation, Derivative Assay, Positive Control, Sequencing, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 is internalized by human B cells. (A) Western blot results analyzing the uptake of C3 by Raji B cells. Cells were treated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met, 10% hiFBS,) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37°C. C3 uptake was investigated by Western blot using the goat polyclonal anti-C3 antibody (Quidel) under non reducing conditions. Data shown are representative of four independent experiments. (B) Western blot results confirming the presence of internalized C3 in the cytoplasm. Cells were incubated with distinct sources of C3 (20% NHS, 200 μg/ml C3, 200 μg/ml C3met) in DGVB++ or EDTA-GVB buffer for 1 h at 37°C. After lysis, cytoplasmic and membrane fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody (Quidel) under non reducing conditions. The purity of distinct cellular fractions was verified using antibodies against B-tubulin (cytoplasmic marker) and Na/K ATPase (membrane marker) under reducing conditions. Results shown are representative of two independent analyzes.

Article Snippet: Antibodies (Abs) used to study C3 expression were the followings: polyclonal goat anti-human C3 (Quidel, #A304) and polyclonal goat anti-human C3 (Calbiochem, #204869).

Techniques: Western Blot, Incubation, Lysis, Marker

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3a in B cells is generated extracellularly by alternative pathway C3-convertases. Western blot results investigating the origin of C3a in Raji B cells. Cells were incubated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37 °C. To analyze complement pathway dependent generation of C3a, sera depleted in C1q (classical pathway), MBL (lectin pathway) or Factor B (alternative pathway) were used. To distinguish intracellular or C3-convertase dependent processing of C3, C3, and C3met were added alone (intracellular cleavage) or in the presence of C3-depleted serum (C3 convertase supplementation). C3 processing and C3a generation were analyzed by Western blot with the goat polyclonal anti-C3 (Quidel) and rabbit anti-C3a (Complement Technologies) antibodies under reducing conditions. Result shown is one representative out of three independent experiments.

Article Snippet: Antibodies (Abs) used to study C3 expression were the followings: polyclonal goat anti-human C3 (Quidel, #A304) and polyclonal goat anti-human C3 (Calbiochem, #204869).

Techniques: Generated, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a enter the nucleus after uptake. (A,B) Western blot results showing presence of C3 and C3a in nuclear compartments of Raji cells. The human B cell line, Raji was incubated with NHS, C3 or C3met as a source of C3 either in EDTA-GVB (A) or in Mg-EGTA (B) buffer for 1 h at 37°C. After lysis, cytoplasmic, soluble nuclear, and chromatin-associated nuclear fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody under non reducing conditions (A) or with the rabbit polyclonal antibody against C3a under reducing condition (B) . The purity of distinct cellular fractions was verified using antibodies against B-actin (cytoplasmic marker), lamin B1 (nuclear marker) and histone H2B (chromatin-associated nuclear marker). Results shown are one representative experiment out of three (A) or four (B) independent analyzes. (C) Representative confocal images showing that AlexaFluor 488 labeled C3 enters the nucleus. 2 × 10 6 Raji cells were incubated with 100 μg/ml C3-AlexaFluor 488 for 30 min at 37°C, fixed and counterstained with DAPI using mounting medium. Representative images are shown from two independent experiments investigating at least 50 cells/analysis.

Article Snippet: Antibodies (Abs) used to study C3 expression were the followings: polyclonal goat anti-human C3 (Quidel, #A304) and polyclonal goat anti-human C3 (Calbiochem, #204869).

Techniques: Western Blot, Incubation, Lysis, Marker, Labeling

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Confirmation of C3-DNA interaction by ChIP sequencing. (A) ChIP-seq identified peaks from anti-C3 immunoprecipitated chromatin samples were classified based on the location (UCSC annotation data) and are shown in the following genome regions: intergenic, introns, downstream 5 Kbp, upstream 5 Kbp, and exons. The interacting DNA elements localized mainly the intergenic and intronic regions. (B) ChIP-seq statistics of identified peaks including peak numbers and genome rate (%). C3 binding to genomic DNA is strongly increased upon stimulation of B cells with the polyclonal activator, CpG oligonucleotide. (C) ChIP-PCR was run on ChIP DNA of CpG-activated samples using primers specific for the ChIP-seq identified regions in IL-7 and L-selectin genes. Enrichment of the specific DNA loci was calculated with the percent input method and illustrated as % input or as fold changes over samples without C3 treatment (two-way ANOVA with Sidak's multiple comparison, * p < 0.05, ** p < 0.01). The values represent the mean ± SEM of four independent experiments.

Article Snippet: Antibodies (Abs) used to study C3 expression were the followings: polyclonal goat anti-human C3 (Quidel, #A304) and polyclonal goat anti-human C3 (Calbiochem, #204869).

Techniques: ChIP-sequencing, Immunoprecipitation, Binding Assay

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Interaction of C3 and its cleavage fragments with histones. Recombinant histone core octamers (H2A/H2B/H3/H4), the linker H1 histone, or alpha-1-antitrypsin (negative control) were immobilized on microtiter plates and incubated with increasing concentrations of C3 (A) , C3b (B) , or C3a (C) . As negative control, BSA was used. Binding was detected either with monoclonal anti-C3d (A,B) or polyclonal anti-C3a (C) antibodies. Background signals obtained from BSA incubation were subtracted from the original values. Data are presented as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to 0 μg/ml protein incubated surfaces (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Upper asterisks indicate statistical significance of H1-, lower asterisks of histone octamer coated surfaces compared to binding to A1AT. (D) gDNA of Raji cells was coated on microtiter plates and AlexaFluor 488-labeled H1 histone binding was monitored in the presence or absence of C3a. As negative control, BSA was used. Background signals obtained on A1AT coated surfaces were subtracted from the original values. Data are presented as relative fluorescent unit (RFU) measured at 525 nm with SD of two independent experiments. Differences with p < 0.05 were considered statistically significant and compared to no C3a treated samples (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01). Upper and lower asterisks indicate statistical significance of H1-AlexaFluor 488 binding in the presence of BSA or C3a (respectively) compared to binding of H1-AlexaFluor 488 alone.

Article Snippet: Antibodies (Abs) used to study C3 expression were the followings: polyclonal goat anti-human C3 (Quidel, #A304) and polyclonal goat anti-human C3 (Calbiochem, #204869).

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Labeling

Journal: Journal of Biological Chemistry

Article Title: Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

doi: 10.1074/jbc.m208420200

Figure Lengend Snippet: FIG. 1. Screen of 33B8 binding deficient PAI-1 mutants. Panels A–C, filter series showing EX(lox) plaques. Panel A, plaques immunore- active with murine monoclonal 33B8; panel B, plaques reactive with rabbit anti-PAI-1 polyclonal antibodies; panel C, a merged image of A and B. The same filter is shown in each case. The dark spots indicate PAI-1antibody complexes, and the circles indicate examples of PAI-1 mutants that are specifically disrupted in 33B8 binding. Panels D and E show immunoblots of cell-free extracts of wild-type and parental PAI-1 mutants selected from the screen. Panel D was probed with 33B8 and panel E with anti-PAI-1 polyclonal antibodies. Panel F shows the inhibitory activity toward high molecular weight uPA of wild-type and each of the PAI-1 parental mutants in cell-free extracts. The inhibitory activity is expressed as the percentage of active PAI-1 protein relative to the total soluble protein of the cell extracts.

Article Snippet: Materials—Murine monoclonal antibodies MA-33B8 (33B8) and MA31C9 (31C9) as well as rabbit polyclonal antibodies directed against human PAI-1 were purchased from Molecular Innovations (Southfield, MI), horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antiserum were from Bio-Rad. tPA was from Genentech (South San Francisco, CA), and uPA was from Molecular Innovations.

Techniques: Binding Assay, Western Blot, Activity Assay, High Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Mapping of a Conformational Epitope on Plasminogen Activator Inhibitor-1 by Random Mutagenesis

doi: 10.1074/jbc.m208420200

Figure Lengend Snippet: FIG. 4. Analysis of site-directed PAI-1 mutants. Panels A and B show immunoblots of cell-free extracts of wild-type and site-directed PAI-1 mutants. Panel A was probed with 33B8 and panel B with anti-PAI-1 polyclonal antibodies. Panel C shows the inhibitory activity toward high molecular weight uPA of wild-type and each of the PAI-1 site-directed mutants in cell-free extracts. The inhibitory activity is expressed as the percentage of functionally active PAI-1 relative to the total soluble protein of the cell extracts.

Article Snippet: Materials—Murine monoclonal antibodies MA-33B8 (33B8) and MA31C9 (31C9) as well as rabbit polyclonal antibodies directed against human PAI-1 were purchased from Molecular Innovations (Southfield, MI), horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antiserum were from Bio-Rad. tPA was from Genentech (South San Francisco, CA), and uPA was from Molecular Innovations.

Techniques: Western Blot, Activity Assay, High Molecular Weight