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Innovagen AB scrambled ll-37
Scrambled Ll 37, supplied by Innovagen AB, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/scrambled+ll-37/ll+37/pm39708316-200-16-17
Average 90 stars, based on 1 article reviews

scrambled ll-37 - by Bioz Stars, 2026-10

90/100 stars

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Article Title: The Human Host Defense Peptide LL-37 Induces Apoptosis in a Calpain- and Apoptosis-Inducing Factor–Dependent Manner Involving Bax Activity
Article Snippet: Scrambled LL-37 (NH2-GLKLRFEFSKIKGEFLKTPEVRFR- DIKLKDNRISVQR-COOH) was synthesized in linear form by Innovagen.

Article Title: The di-leucine motif in the host defense peptide LL-37 is essential for initiation of autophagy in human macrophages.
Article Snippet: Article

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M.tb infection and cellular activation by IFNγ enhance cell surface GAPDH, LL-37 binding, and internalization. a–c Infection with M.tb enhances total and cell surface GAPDH recruitment but does not alter the expression of cell surface P2X7 receptor on macrophages. a C57BL/6 mice were injected i.p. with 1 × 10 7 M.tb H37Rv-GFP bacilli per mouse. Peritoneal cells were harvested from control and infected mice after 48 h and cell surface P2X7 expression on infected cells was quantified by flow cytometry. Data are shown as representative overlay histogram and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 3 (NS, p > 0.5 and overlay is representative of three independent experiments) ( b ) GAPDH activity in whole cell lysate of infected cell cultures was quantified using KDalert TM GAPDH Assay Kit (Ambion) and was found to be significantly increased. Data are presented as GAPDH activity/mg protein of cell lysate ± SD ( n = 5, *** p < 0.001). c–e C57BL/6 mice were injected i.p. with 1 × 10 7 M.tb H37Rv-mCherry bacilli/mouse. Peritoneal cells were harvested from control and infected mice after 48 h and cell surface GAPDH level on infected cells was quantified by flow cytometry using anti-GAPDH antibody ( c ). Cell surface binding ( d ) and uptake ( e ) of LL-37-FAM (10 μg/mL) by infected cells were assessed by flow cytometry and found to be significantly enhanced. Data are shown as representative overlay histogram and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 2, *** p < 0.001, and overlay is representative of two independent experiments). In comparison to control wild-type cells, cells wherein GAPDH had been knocked down demonstrated a significant decrease in cell surface expression of GAPDH ( f ) which correlated with significant decrease in LL-37 binding ( g ) as well as diminished uptake of the peptide ( h ). Data in inset are presented as mean fluorescence intensity (MFI ± SEM) (*** p < 0.0001, n = 10 4 cells and overlay is representative of three independent experiments). IFNγ treatment resulted in significant increase in total cellular GAPDH ( i ). The increase in total cellular GAPDH by IFNγ treatment also resulted in significant increase in GAPDH recruitment to cell surface ( j ), enhanced LL-37 binding ( k ), and its internalization ( l ). Data are shown as representative overlay histogram and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 3 (*** p < 0.0001 and is representative of three independent experiments). For gating strategy utilized in flow cytometry experiments, please see online suppl. Fig. S3.

Journal: Journal of Innate Immunity

Article Title: Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell

doi: 10.1159/000530083

Figure Lengend Snippet: M.tb infection and cellular activation by IFNγ enhance cell surface GAPDH, LL-37 binding, and internalization. a–c Infection with M.tb enhances total and cell surface GAPDH recruitment but does not alter the expression of cell surface P2X7 receptor on macrophages. a C57BL/6 mice were injected i.p. with 1 × 10 7 M.tb H37Rv-GFP bacilli per mouse. Peritoneal cells were harvested from control and infected mice after 48 h and cell surface P2X7 expression on infected cells was quantified by flow cytometry. Data are shown as representative overlay histogram and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 3 (NS, p > 0.5 and overlay is representative of three independent experiments) ( b ) GAPDH activity in whole cell lysate of infected cell cultures was quantified using KDalert TM GAPDH Assay Kit (Ambion) and was found to be significantly increased. Data are presented as GAPDH activity/mg protein of cell lysate ± SD ( n = 5, *** p < 0.001). c–e C57BL/6 mice were injected i.p. with 1 × 10 7 M.tb H37Rv-mCherry bacilli/mouse. Peritoneal cells were harvested from control and infected mice after 48 h and cell surface GAPDH level on infected cells was quantified by flow cytometry using anti-GAPDH antibody ( c ). Cell surface binding ( d ) and uptake ( e ) of LL-37-FAM (10 μg/mL) by infected cells were assessed by flow cytometry and found to be significantly enhanced. Data are shown as representative overlay histogram and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 2, *** p < 0.001, and overlay is representative of two independent experiments). In comparison to control wild-type cells, cells wherein GAPDH had been knocked down demonstrated a significant decrease in cell surface expression of GAPDH ( f ) which correlated with significant decrease in LL-37 binding ( g ) as well as diminished uptake of the peptide ( h ). Data in inset are presented as mean fluorescence intensity (MFI ± SEM) (*** p < 0.0001, n = 10 4 cells and overlay is representative of three independent experiments). IFNγ treatment resulted in significant increase in total cellular GAPDH ( i ). The increase in total cellular GAPDH by IFNγ treatment also resulted in significant increase in GAPDH recruitment to cell surface ( j ), enhanced LL-37 binding ( k ), and its internalization ( l ). Data are shown as representative overlay histogram and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 3 (*** p < 0.0001 and is representative of three independent experiments). For gating strategy utilized in flow cytometry experiments, please see online suppl. Fig. S3.

Article Snippet: To determine the signaling pathway for LL-37 induction or its internalization, cells were treated with 100 n m KN-62 (1-(N,O-bis(5-Isoquinolinesulfonyl)-N-methyl- l -tyrosyl)-4-phenylpiperazine), a P2RX7 receptor inhibitor for 4 h (Calbiochem), p38 mitogen-activated protein kinase (MAPK) inhibitor SB0502880 (Sigma) for 1 h, cholesterol synthesis inhibitor mevinolin and methyl-β-cyclodextrin (to disrupt membrane rafts) (Sigma) for 4 h, bafilomycin 100 nm alone for 2 h or before treatment with LL-37 (AnaSpec #AS-61302) or scrambled LL-37 (AnaSpec #AS-63708).

Techniques: Infection, Activation Assay, Binding Assay, Expressing, Injection, Flow Cytometry, Fluorescence, Activity Assay

Surface GAPDH moonlights as an LL-37 receptor, internalizing it in a lipid raft-dependent process. a–c THP-1 empty vector and GAPDH knockdown cells were treated with IFNγ. Elevation of both cell surface GAPDH ( a ) and P2×7 levels ( b ) was confirmed by flow cytometry. Internalization of LL-37-FAM into these cells was quantified using flow cytometry and found to be sensitive to surface recruitment of GAPDH. c All data are presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 3 (*** p < 0.005, *** p < 0.0001, *** p < 0.001 and overlay is representative of three independent experiments). d , e Treatment with LL-37 results in elevated cytosolic Ca ++ levels. PMA-activated THP-1 cells were treated with LL-37 peptide (10 μg/mL) for 12 h and stained with 2 μ m fluo-3-AM for 30 min. Cells were visualized by confocal microscopy, representative image scale bars, 10 μm. d Cellular fluo-3-AM fluorescence was also quantified using flow cytometry, Ca ++ signal is not enhanced in GAPDH knockdown THP-1 cells. Flow cytometry data are presented as mean fluorescence intensity (MFI ± SEM), from 10 4 cells for n = 3 *** p < 0.0001 ( e ). f–h Inhibition of CaMK II with KN-62 results in inhibition of LL-37 uptake into J774 wild-type cells ( f ). This is accompanied with a decrease in cell surface GAPDH ( g ) and intracellular Ca 2+ levels ( h ). Data are shown as representative overlay histogram and are representative of three independent experiments and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells with at least *** p < 0.005, n = 3. GAPDH and LL-37-FAM co-localize on macrophage plasma membrane ( i ). J774 cells were stained with polyclonal mouse anti-GAPDH antibody for 1 hour at 4°C; subsequently, after extensive washing, cells were incubated with anti-mouse Alexa 568 and 5 μg of LL-37-FAM. After 3 rinses, cells were shifted to 37°C for 20 min to allow for internalization of surface-bound molecules. Cells were then fixed and visualized under a confocal microscope. Scale bar, 5 μm. Pearson’s correlation coefficient was calculated to evaluate the extent of co-localization (*** p < 0.0001, n = 10). Co-IP assay revealed LL-37 interaction with membrane GAPDH (for detailed explanation of protocol, see online supplementary methods) ( j ). Disruption of membrane rafts on J774 plasma membrane by treatment with mevinolin (MEV) and methyl-β-cyclodextrin (MβCD) confirmed by significant loss of cholera toxin-B Alexa 647 binding using flow cytometry ( k ). Results in box plot are presented as MFI ± SEM from 10 4 cells (*** p < 0.001, n = 2). Binding of LL-37 and co-localization with GAPDH in lipid rafts was confirmed by confocal microscopy ( l , upper panel). Disruption of rafts resulted in decrease of, GAPDH signal as well as LL-37 binding ( l , lower panel). m–o Treatment of cells with IFNγ enhances GAPDH on membrane ( m ) which correlates with enhanced LL-37 binding ( n ) and uptake ( o ); disruption of rafts depletes surface GAPDH ( m ) with cells binding and internalizing significantly less of LL-37 ( n , o ). GAPDH expression, LL-37 binding, and internalization were evaluated by flow cytometry after IFNγ treatment and lipid raft disruption. Flow cytometry results are presented in box plot as MFI ± SEM from 10 4 cells (*** p < 0.001, n = 3). For gating strategy utilized in flow cytometry experiments, please see online supplementary Fig. S3. CaMK II, calmodulin-dependent kinase type II. IP, immunoprecipitation; IB, immunoblotting.

Journal: Journal of Innate Immunity

Article Title: Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell

doi: 10.1159/000530083

Figure Lengend Snippet: Surface GAPDH moonlights as an LL-37 receptor, internalizing it in a lipid raft-dependent process. a–c THP-1 empty vector and GAPDH knockdown cells were treated with IFNγ. Elevation of both cell surface GAPDH ( a ) and P2×7 levels ( b ) was confirmed by flow cytometry. Internalization of LL-37-FAM into these cells was quantified using flow cytometry and found to be sensitive to surface recruitment of GAPDH. c All data are presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells for n = 3 (*** p < 0.005, *** p < 0.0001, *** p < 0.001 and overlay is representative of three independent experiments). d , e Treatment with LL-37 results in elevated cytosolic Ca ++ levels. PMA-activated THP-1 cells were treated with LL-37 peptide (10 μg/mL) for 12 h and stained with 2 μ m fluo-3-AM for 30 min. Cells were visualized by confocal microscopy, representative image scale bars, 10 μm. d Cellular fluo-3-AM fluorescence was also quantified using flow cytometry, Ca ++ signal is not enhanced in GAPDH knockdown THP-1 cells. Flow cytometry data are presented as mean fluorescence intensity (MFI ± SEM), from 10 4 cells for n = 3 *** p < 0.0001 ( e ). f–h Inhibition of CaMK II with KN-62 results in inhibition of LL-37 uptake into J774 wild-type cells ( f ). This is accompanied with a decrease in cell surface GAPDH ( g ) and intracellular Ca 2+ levels ( h ). Data are shown as representative overlay histogram and are representative of three independent experiments and box plot in the inset is presented as mean fluorescence intensity (MFI ± SEM) from 10 4 cells with at least *** p < 0.005, n = 3. GAPDH and LL-37-FAM co-localize on macrophage plasma membrane ( i ). J774 cells were stained with polyclonal mouse anti-GAPDH antibody for 1 hour at 4°C; subsequently, after extensive washing, cells were incubated with anti-mouse Alexa 568 and 5 μg of LL-37-FAM. After 3 rinses, cells were shifted to 37°C for 20 min to allow for internalization of surface-bound molecules. Cells were then fixed and visualized under a confocal microscope. Scale bar, 5 μm. Pearson’s correlation coefficient was calculated to evaluate the extent of co-localization (*** p < 0.0001, n = 10). Co-IP assay revealed LL-37 interaction with membrane GAPDH (for detailed explanation of protocol, see online supplementary methods) ( j ). Disruption of membrane rafts on J774 plasma membrane by treatment with mevinolin (MEV) and methyl-β-cyclodextrin (MβCD) confirmed by significant loss of cholera toxin-B Alexa 647 binding using flow cytometry ( k ). Results in box plot are presented as MFI ± SEM from 10 4 cells (*** p < 0.001, n = 2). Binding of LL-37 and co-localization with GAPDH in lipid rafts was confirmed by confocal microscopy ( l , upper panel). Disruption of rafts resulted in decrease of, GAPDH signal as well as LL-37 binding ( l , lower panel). m–o Treatment of cells with IFNγ enhances GAPDH on membrane ( m ) which correlates with enhanced LL-37 binding ( n ) and uptake ( o ); disruption of rafts depletes surface GAPDH ( m ) with cells binding and internalizing significantly less of LL-37 ( n , o ). GAPDH expression, LL-37 binding, and internalization were evaluated by flow cytometry after IFNγ treatment and lipid raft disruption. Flow cytometry results are presented in box plot as MFI ± SEM from 10 4 cells (*** p < 0.001, n = 3). For gating strategy utilized in flow cytometry experiments, please see online supplementary Fig. S3. CaMK II, calmodulin-dependent kinase type II. IP, immunoprecipitation; IB, immunoblotting.

Techniques: Plasmid Preparation, Flow Cytometry, Fluorescence, Staining, Confocal Microscopy, Inhibition, Incubation, Microscopy, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Immunoprecipitation, Western Blot

$LL-37 peptide interacts with cell surface GAPDH and internalizes into endocytic vesicles. a Endocytic co-localization of membrane GAPDH and LL-37-FAM. J774 cells were incubated with polyclonal mouse anti-GAPDH antibody, followed by anti-mouse-A568 and 5 μg of LL-37-FAM for 1 hour at 4°C for staining cell surface. After rinsing ×3 with buffer, cells were brought to 37°C to allow internalization of surface-labeled molecules for 20 min. Cells were then fixed and visualized in a confocal microscope. Scale bars, 5 μm. Pearson’s correlation coefficient was calculated to evaluate the co-localization of GAPDH and LL-37. Cells where primary GAPDH antibody was omitted were used as control background (*** p < 0.0001, n = 10 cells). b Acceptor photobleaching FRET reveals interaction between LL-37 and GAPDH co-localized in endocytic vesicles. c Comparison of FRET efficiencies (*** p < 0.0001, n = 30). d For interaction studies by Co-IP, 10 μg of LL-37 was allowed to bind onto the surface of J774 macrophages. Subsequently, the cells were allowed to internalize LL-37 or scrambled peptide (used as a control) for 30 min to allow for its trafficking into endosomes. Subsequently, cells were washed thoroughly with 2% BSA in chilled PBS. Endosomal fractions (also see online suppl. Fig. S1E) were prepared as described in material and methods. Fractions were lysed and LL-37 was pulled down using anti-LL-37 antibody conjugated onto magnabeads. The pull-down fraction was processed for western blotting. Membrane blots were probed with anti-GAPDH antibody and anti-LL-37 antibody. e Affinity analysis of GAPDH-LL-37 interaction by MST; data are plotted as dose response curve fit. LL-37 concentrations are plotted on the x-axis in (M). Kd was computed to be 315 ± 40 n m ; representative graph from triplicate set of experiment is presented. f Scrambled peptide was used as negative control (inset e) . GAPDH interacts with both human LL-37 peptide and mCRAMP peptide. MST revealed a Kd of 472 nm for interaction between GAPDH and mCRAMP peptide. IP, immunoprecipitation; IB, immunoblotting.$

Journal: Journal of Innate Immunity

Article Title: Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell

doi: 10.1159/000530083

Figure Lengend Snippet: LL-37 peptide interacts with cell surface GAPDH and internalizes into endocytic vesicles. a Endocytic co-localization of membrane GAPDH and LL-37-FAM. J774 cells were incubated with polyclonal mouse anti-GAPDH antibody, followed by anti-mouse-A568 and 5 μg of LL-37-FAM for 1 hour at 4°C for staining cell surface. After rinsing ×3 with buffer, cells were brought to 37°C to allow internalization of surface-labeled molecules for 20 min. Cells were then fixed and visualized in a confocal microscope. Scale bars, 5 μm. Pearson’s correlation coefficient was calculated to evaluate the co-localization of GAPDH and LL-37. Cells where primary GAPDH antibody was omitted were used as control background (*** p < 0.0001, n = 10 cells). b Acceptor photobleaching FRET reveals interaction between LL-37 and GAPDH co-localized in endocytic vesicles. c Comparison of FRET efficiencies (*** p < 0.0001, n = 30). d For interaction studies by Co-IP, 10 μg of LL-37 was allowed to bind onto the surface of J774 macrophages. Subsequently, the cells were allowed to internalize LL-37 or scrambled peptide (used as a control) for 30 min to allow for its trafficking into endosomes. Subsequently, cells were washed thoroughly with 2% BSA in chilled PBS. Endosomal fractions (also see online suppl. Fig. S1E) were prepared as described in material and methods. Fractions were lysed and LL-37 was pulled down using anti-LL-37 antibody conjugated onto magnabeads. The pull-down fraction was processed for western blotting. Membrane blots were probed with anti-GAPDH antibody and anti-LL-37 antibody. e Affinity analysis of GAPDH-LL-37 interaction by MST; data are plotted as dose response curve fit. LL-37 concentrations are plotted on the x-axis in (M). Kd was computed to be 315 ± 40 n m ; representative graph from triplicate set of experiment is presented. f Scrambled peptide was used as negative control (inset e) . GAPDH interacts with both human LL-37 peptide and mCRAMP peptide. MST revealed a Kd of 472 nm for interaction between GAPDH and mCRAMP peptide. IP, immunoprecipitation; IB, immunoblotting.

Techniques: Incubation, Staining, Labeling, Microscopy, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Immunoprecipitation

GAPDH is essential for LL-37-mediated induction of autophagy. PMA-activated THP-1 empty vector and GAPDH knockdown cells were infected with H37Rv (MOI: 1:5) and treated with LL-37 peptide for 24 h either alone ( a , b ) or in the presence of bafilomycin A1 ( c , d ). Cells were processed for preparation of total cell lysate and analyzed for autophagy markers LC3-I to LC3-II conversion and p62 by Western blotting. Images were quantified by ImageJ software. Changes in LC3-II protein levels are illustrated by LC3-II/LC3-I ratios in bar graphs ( b , d ). Data are plotted as mean ± SD from 2 independent experiments (*** p < 0.0001, NS: p > 0.05). GAPDH mediates upregulation of autophagy genes ( e , f ). Cells were processed for RNA isolation and analyzed for expression of autophagy pathway genes ATG5 ( e ) and ATG7 ( f ) by real-time qPCR. Data are presented as fold change (*** p < 0.001, n = 2). LL-37-mediated fusion of M.tb containing phagosomes with lysosomes is hampered in GAPDH knockdown cells ( g , h ). PMA-activated THP-1 empty vector and GAPDH knockdown cells were infected with H37Rv expressing EGFP (MOI: 1:5) and treated with 1 μg/mL of LL-37 peptide for 24 h. Lysosomes were visualized using LysoTracker ® Red and imaged using a confocal microscope. Scale bars, 10 μm. Co-localization events of EGFP expressing bacteria and LysoTracker red dye labeled vesicles were counted ( g ). Percentage co-localization or fusion of M.tb H37Rv EGFP with lysosomes was calculated from randomly acquired images ( h ) (*** p < 0.001, n = 100). GAPDH knockdown cells do not demonstrate LL-37-mediated anti-mycobacterial activity ( i ). Infected cells were analyzed for bacterial survival assay. Cells were processed for CFU measurement at 0, 24, and 48 h post-addition of LL-37 at 50 μg/mL. 7H11 plates were analyzed after 21 days and colonies were counted. Results are expressed as mean CFU/mL cell lysate at indicated times (** p < 0.001, n = 3). LL-37 anti-mycobacterial functions involve p38 MAPK signaling ( j–l ). PMA-activated THP-1 empty vector and GAPDH knockdown cells were infected with M.tb H37Rv (MOI: 1:5) and pretreated for 3 h with p38 inhibitor (SB023080) followed by treatment with LL-37 peptide for 30 min to check for activation of p-p38 MAPK by Western blot ( j ) or 24 h for evaluation of autophagy induction by probing for autophagy marker LC-3B ( k ). Western blot images were quantified by ImageJ software. Changes in LC3-II protein levels are illustrated by LC3-II/LC3-I ratios in bar graphs ( l ). Data are plotted as mean ± SD from 3 independent experiments (*** p < 0.001 as compared to control or as indicated). MOI, multiplicity of infection.

Journal: Journal of Innate Immunity

Article Title: Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell

doi: 10.1159/000530083

Figure Lengend Snippet: GAPDH is essential for LL-37-mediated induction of autophagy. PMA-activated THP-1 empty vector and GAPDH knockdown cells were infected with H37Rv (MOI: 1:5) and treated with LL-37 peptide for 24 h either alone ( a , b ) or in the presence of bafilomycin A1 ( c , d ). Cells were processed for preparation of total cell lysate and analyzed for autophagy markers LC3-I to LC3-II conversion and p62 by Western blotting. Images were quantified by ImageJ software. Changes in LC3-II protein levels are illustrated by LC3-II/LC3-I ratios in bar graphs ( b , d ). Data are plotted as mean ± SD from 2 independent experiments (*** p < 0.0001, NS: p > 0.05). GAPDH mediates upregulation of autophagy genes ( e , f ). Cells were processed for RNA isolation and analyzed for expression of autophagy pathway genes ATG5 ( e ) and ATG7 ( f ) by real-time qPCR. Data are presented as fold change (*** p < 0.001, n = 2). LL-37-mediated fusion of M.tb containing phagosomes with lysosomes is hampered in GAPDH knockdown cells ( g , h ). PMA-activated THP-1 empty vector and GAPDH knockdown cells were infected with H37Rv expressing EGFP (MOI: 1:5) and treated with 1 μg/mL of LL-37 peptide for 24 h. Lysosomes were visualized using LysoTracker ® Red and imaged using a confocal microscope. Scale bars, 10 μm. Co-localization events of EGFP expressing bacteria and LysoTracker red dye labeled vesicles were counted ( g ). Percentage co-localization or fusion of M.tb H37Rv EGFP with lysosomes was calculated from randomly acquired images ( h ) (*** p < 0.001, n = 100). GAPDH knockdown cells do not demonstrate LL-37-mediated anti-mycobacterial activity ( i ). Infected cells were analyzed for bacterial survival assay. Cells were processed for CFU measurement at 0, 24, and 48 h post-addition of LL-37 at 50 μg/mL. 7H11 plates were analyzed after 21 days and colonies were counted. Results are expressed as mean CFU/mL cell lysate at indicated times (** p < 0.001, n = 3). LL-37 anti-mycobacterial functions involve p38 MAPK signaling ( j–l ). PMA-activated THP-1 empty vector and GAPDH knockdown cells were infected with M.tb H37Rv (MOI: 1:5) and pretreated for 3 h with p38 inhibitor (SB023080) followed by treatment with LL-37 peptide for 30 min to check for activation of p-p38 MAPK by Western blot ( j ) or 24 h for evaluation of autophagy induction by probing for autophagy marker LC-3B ( k ). Western blot images were quantified by ImageJ software. Changes in LC3-II protein levels are illustrated by LC3-II/LC3-I ratios in bar graphs ( l ). Data are plotted as mean ± SD from 3 independent experiments (*** p < 0.001 as compared to control or as indicated). MOI, multiplicity of infection.

Techniques: Plasmid Preparation, Infection, Western Blot, Software, Isolation, Expressing, Microscopy, Labeling, Activity Assay, Clonogenic Cell Survival Assay, Activation Assay, Marker

LL-37 (A), or lysozyme (B) were added to pyrene labeled (10% labeling ratio) 2 µM MgATP-G-actin and the polymerization was followed by increase in pyrene fluorescence. Presented data are representative of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: LL-37 (A), or lysozyme (B) were added to pyrene labeled (10% labeling ratio) 2 µM MgATP-G-actin and the polymerization was followed by increase in pyrene fluorescence. Presented data are representative of three independent experiments.

Article Snippet: LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) peptide and scrambled LL-37 (sLL-37) peptide (GLKLRFEFSKIKGEFLKTPEVRFRDIKLKDNRISVQR) were purchased from (Genemed Synthesis Inc., San Antonio,TX ).

Techniques: Labeling, Fluorescence

4 µM LL-37 (A) or lysozyme (B) were added (simultaneously with NaCl or MgCl 2 ) to pyrene labeled (10% labeling ratio) 2 µM MgATP-G-actin and the polymerization was followed by an increase in pyrene fluorescence. Presented data are representative of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: 4 µM LL-37 (A) or lysozyme (B) were added (simultaneously with NaCl or MgCl 2 ) to pyrene labeled (10% labeling ratio) 2 µM MgATP-G-actin and the polymerization was followed by an increase in pyrene fluorescence. Presented data are representative of three independent experiments.

Techniques: Labeling, Fluorescence

( A ) Kinetics of LL-37-induced bundle formation: LL-37 (2–8 µM) was added to MgF-actin (2 µM) and bundle formation was followed as an increase in light scattering at 350 nm. Presented data are representative of three independent experiments. ( B ) Extent of LL-37 and lysozyme induced bundle formation: LL-37 or lysozyme (0.5–9 µM) were added to Mg-F-actin (4 µM). Samples were centrifuged at 20,800×g, for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. ( C ) Extent of LL-37 and scrambled LL-37 induced bundle formation: LL-37 or scrambled LL-37 (2–14 µM) were added to MgF-actin (4 µM) and bundle formation was measured as in (B). The presented data are mean and standard deviation of three independent experiments in (B) and (C).

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: ( A ) Kinetics of LL-37-induced bundle formation: LL-37 (2–8 µM) was added to MgF-actin (2 µM) and bundle formation was followed as an increase in light scattering at 350 nm. Presented data are representative of three independent experiments. ( B ) Extent of LL-37 and lysozyme induced bundle formation: LL-37 or lysozyme (0.5–9 µM) were added to Mg-F-actin (4 µM). Samples were centrifuged at 20,800×g, for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. ( C ) Extent of LL-37 and scrambled LL-37 induced bundle formation: LL-37 or scrambled LL-37 (2–14 µM) were added to MgF-actin (4 µM) and bundle formation was measured as in (B). The presented data are mean and standard deviation of three independent experiments in (B) and (C).

Techniques: SDS Page, Standard Deviation

( A ) 6 µM lysozyme or LL-37 were added to Mg-F-actin (4 µM). ( B ) 10 µM LL-37 or sLL-37 were added to 4 µM MgF-actin After 10 min incubation at room temperature samples were centrifuged at 20,800×g for 8 min. The supernatants were analyzed by SDS-PAGE and densitometry. The presented data are mean and standard deviation of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: ( A ) 6 µM lysozyme or LL-37 were added to Mg-F-actin (4 µM). ( B ) 10 µM LL-37 or sLL-37 were added to 4 µM MgF-actin After 10 min incubation at room temperature samples were centrifuged at 20,800×g for 8 min. The supernatants were analyzed by SDS-PAGE and densitometry. The presented data are mean and standard deviation of three independent experiments.

Techniques: Incubation, SDS Page, Standard Deviation

µM LL-37 on subtilisin digestion of CaATP-G-actin, MgATP-G-actin and Mg-F-actin. 8 µM CaATP-G-actin or 8 µM MgATP-G-actin were digested by 4 µg/ml subtilisin for 2 min, MgF-actin (8 µM) was digested by 20 µg/ml subtilisin for 30 min in the presence or absence of 6 µM LL-37. Digestions were carried out at 22°C and quenched by 1 mM PMSF. Samples were analyzed by 12% SDS-PAGE and densitometry. Gel insert shows representative actin bands: (A), actin only; (B), CaATP-G-actin, subtilisin, no LL-37; (C), CaATP-G-actin,subtilisin and LL-37, (D) MgATP-G-actin, subtilisin, no LL-37, (E) MgATP-G-actin, subtilisin and LL-37, (F) Mg-F-actin, subtilisin, no LL-37, (G) Mg-F-actin, subtilisin and LL-37, (H) Mg-F-actin, 200 mM NaCl, subtilisin, no LL-37, (I) Mg-F-actin, 200 mM NaCl, subtilisin and LL-37. The presented quantitation data are mean and standard deviation of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: µM LL-37 on subtilisin digestion of CaATP-G-actin, MgATP-G-actin and Mg-F-actin. 8 µM CaATP-G-actin or 8 µM MgATP-G-actin were digested by 4 µg/ml subtilisin for 2 min, MgF-actin (8 µM) was digested by 20 µg/ml subtilisin for 30 min in the presence or absence of 6 µM LL-37. Digestions were carried out at 22°C and quenched by 1 mM PMSF. Samples were analyzed by 12% SDS-PAGE and densitometry. Gel insert shows representative actin bands: (A), actin only; (B), CaATP-G-actin, subtilisin, no LL-37; (C), CaATP-G-actin,subtilisin and LL-37, (D) MgATP-G-actin, subtilisin, no LL-37, (E) MgATP-G-actin, subtilisin and LL-37, (F) Mg-F-actin, subtilisin, no LL-37, (G) Mg-F-actin, subtilisin and LL-37, (H) Mg-F-actin, 200 mM NaCl, subtilisin, no LL-37, (I) Mg-F-actin, 200 mM NaCl, subtilisin and LL-37. The presented quantitation data are mean and standard deviation of three independent experiments.

Techniques: SDS Page, Quantitation Assay, Standard Deviation

Biacore sensorgram [fit (solid) and experimental (dashed)] showing the interactions between LL-37 (A) or scrambled LL-37 (B) and actin, using series of concentrations of 0–500 nM (A), 0–2500 nM (B). Calculation for sLL-37 (B) were performed using a steady state affinity model based on the sensorgram shown as an insert in B. Presented data are representative of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: Biacore sensorgram [fit (solid) and experimental (dashed)] showing the interactions between LL-37 (A) or scrambled LL-37 (B) and actin, using series of concentrations of 0–500 nM (A), 0–2500 nM (B). Calculation for sLL-37 (B) were performed using a steady state affinity model based on the sensorgram shown as an insert in B. Presented data are representative of three independent experiments.

Techniques:

Dissociation constants of LL-37 bound to immobilized G-actin using the BIACORE 3000 system.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: Dissociation constants of LL-37 bound to immobilized G-actin using the BIACORE 3000 system.

Techniques:

5 µM DNase1 or cofilin were added to 4 µM Mg-F-actin bundles (bundled by 4–9 µM LL-37) in the presence or absence of 100 mM NaCl. Following 30 min incubation the samples were centrifuged at 20,800×g for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. ( A ) Dissociation by DNase1. ( B ) Dissociation by cofilin. The presented data are mean and standard deviation of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: 5 µM DNase1 or cofilin were added to 4 µM Mg-F-actin bundles (bundled by 4–9 µM LL-37) in the presence or absence of 100 mM NaCl. Following 30 min incubation the samples were centrifuged at 20,800×g for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. ( A ) Dissociation by DNase1. ( B ) Dissociation by cofilin. The presented data are mean and standard deviation of three independent experiments.

Techniques: Incubation, SDS Page, Standard Deviation

LL-37 (9 µM) was added to 4 µM MgF-actin (thin arrow), inducing immediate bundling. This was followed by the addition of 6 µM DNase1, 6 µM cofilin, or 200 mM NaCl (thick arrow). Bundling and dissociation were followed as an increase and decrease in light scattering, respectively, measured at 450 nm. Presented data are representative of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: LL-37 (9 µM) was added to 4 µM MgF-actin (thin arrow), inducing immediate bundling. This was followed by the addition of 6 µM DNase1, 6 µM cofilin, or 200 mM NaCl (thick arrow). Bundling and dissociation were followed as an increase and decrease in light scattering, respectively, measured at 450 nm. Presented data are representative of three independent experiments.

Techniques:

LL-37 (9 µM) was added to 2 µM Mg-F-actin. Following a 10 minute incubation, 6 µM DNase1 or 9 µM cofilin were added. After 30 min incubation the samples were divided into two pools and centrifuged at 20,800×g or 352,271× g for 8 and 60 min, respectively. The supernatants were analyzed by SDS-PAGE and densitometry. Monomer (G-actin) separated from actin filaments (F-actin) by one hour 352,271× g centrifugation. Bundled F-actin was separated from unbundled F-actin by low speed (20,800× g for 8 min) centrifugation. Thus, the three actin forms (G-, unbundled F- and bundled F-actin) were separated from each other by low and high speed centrifugations. Addition of 6 µM DNase1 and 9 µM cofilin to 4 µM Mg-F-actin (bundled by 9 µM LL-37) promoted disappearance of bundles at 100% and 73%, respectively. The presented data are mean and standard deviation of three independent experiments. Gel insert shows representative actin bands: (A) MgF-actin, pre-spin; (B) MgF-actin low speed; (C) MgF-actin high speed centrifugation; (D) MgF-actin, LL-37 pre-spin; (E) MgF-actin, LL-37 low speed; (F) MgF-actin, LL-37 high speed centrifugation; (G) MgF-actin, LL-37, DNase1 pre-spin; (H) MgF-actin, LL-37, DNase1 low speed; (I) MgF-actin, LL-37, DNase1 high speed centrifugation; (J) MgF-actin, LL-37, cofilin pre-spin; (K) MgF-actin, LL-37, cofilin low speed; (L) MgF-actin, LL-37, cofilin high speed centrifugation.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: LL-37 (9 µM) was added to 2 µM Mg-F-actin. Following a 10 minute incubation, 6 µM DNase1 or 9 µM cofilin were added. After 30 min incubation the samples were divided into two pools and centrifuged at 20,800×g or 352,271× g for 8 and 60 min, respectively. The supernatants were analyzed by SDS-PAGE and densitometry. Monomer (G-actin) separated from actin filaments (F-actin) by one hour 352,271× g centrifugation. Bundled F-actin was separated from unbundled F-actin by low speed (20,800× g for 8 min) centrifugation. Thus, the three actin forms (G-, unbundled F- and bundled F-actin) were separated from each other by low and high speed centrifugations. Addition of 6 µM DNase1 and 9 µM cofilin to 4 µM Mg-F-actin (bundled by 9 µM LL-37) promoted disappearance of bundles at 100% and 73%, respectively. The presented data are mean and standard deviation of three independent experiments. Gel insert shows representative actin bands: (A) MgF-actin, pre-spin; (B) MgF-actin low speed; (C) MgF-actin high speed centrifugation; (D) MgF-actin, LL-37 pre-spin; (E) MgF-actin, LL-37 low speed; (F) MgF-actin, LL-37 high speed centrifugation; (G) MgF-actin, LL-37, DNase1 pre-spin; (H) MgF-actin, LL-37, DNase1 low speed; (I) MgF-actin, LL-37, DNase1 high speed centrifugation; (J) MgF-actin, LL-37, cofilin pre-spin; (K) MgF-actin, LL-37, cofilin low speed; (L) MgF-actin, LL-37, cofilin high speed centrifugation.

Techniques: Incubation, SDS Page, Centrifugation, Standard Deviation

Mg-F-actin (4 µM) was bundled by 9 µM LL-37 or lysozyme in the presence of 100 mM NaCl. After 10 min of incubation, DNase1 or cofilin were added in increasing concentrations to bundled actin. After 30 min of incubation, the samples were centrifuged at 20,800× g for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. ( A ) Unbundling of LL-37-induced Mg-F-actin bundles. ( B ) Unbundling of lysozyme-induced Mg-F-actin bundles. The presented data are mean and standard deviation of three independent experiments.

Journal: PLoS ONE

Article Title: LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

doi: 10.1371/journal.pone.0050078

Figure Lengend Snippet: Mg-F-actin (4 µM) was bundled by 9 µM LL-37 or lysozyme in the presence of 100 mM NaCl. After 10 min of incubation, DNase1 or cofilin were added in increasing concentrations to bundled actin. After 30 min of incubation, the samples were centrifuged at 20,800× g for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. ( A ) Unbundling of LL-37-induced Mg-F-actin bundles. ( B ) Unbundling of lysozyme-induced Mg-F-actin bundles. The presented data are mean and standard deviation of three independent experiments.

Techniques: Incubation, SDS Page, Standard Deviation

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scrambled ll-37 (Innovagen AB)

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