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    Structured Review

    Thermo Fisher leupeptin
    RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml <t>leupeptin.</t> Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .
    Leupeptin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Chaperone-independent peripheral quality control of CFTR by RFFL E3 ligase"

    Article Title: Chaperone-independent peripheral quality control of CFTR by RFFL E3 ligase

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.02.001

    RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml leupeptin. Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .
    Figure Legend Snippet: RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml leupeptin. Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .

    Techniques Used: Fluorescence, Expressing, Transfection, Immunostaining, Labeling, Marker, Stable Transfection, Periodic Counter-current Chromatography

    2) Product Images from "Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons"

    Article Title: Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons

    Journal: Scientific Reports

    doi: 10.1038/srep05715

    EBSS and E + L treatments induced AVs in primary cortical neurons. (a) LC3-II was analyzed by western blotting in the cell lysate prepared from EBSS (E), leupeptin (L) and EBSS + leupeptin (E + L) treated neurons. The LC3-II levels were compared to those of actin. For the analysis of LC3-II, 10 μg of cell lysate were loaded (n = 6). Values represent mean ± s.e.m.; *p
    Figure Legend Snippet: EBSS and E + L treatments induced AVs in primary cortical neurons. (a) LC3-II was analyzed by western blotting in the cell lysate prepared from EBSS (E), leupeptin (L) and EBSS + leupeptin (E + L) treated neurons. The LC3-II levels were compared to those of actin. For the analysis of LC3-II, 10 μg of cell lysate were loaded (n = 6). Values represent mean ± s.e.m.; *p

    Techniques Used: Western Blot

    EBSS, leupeptin and EBSS + leupeptin increased the secretion of endogenous tau by primary cortical neurons. (a) In normal conditions (CTL), one tau-positive band at 52 kDa was detected in the culture medium by an antibody revealing total tau (A0024). A similar pattern was observed when neurons were treated with leupeptin (L). When neurons were treated either with EBSS (E) or EBSS + leupeptin (E + L), three tau-positive bands at 47, 52 and 57 kDa were detected in the culture medium. In the cell lysate of control neurons, two tau-positive bands at 52 and 57 kDa were revealed by the anti-tau antibody A0024 (n = 6). (b) Quantification of total tau in culture medium by densitometry upon EBSS, leupeptin and E + L treatments (n = 6). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; *p
    Figure Legend Snippet: EBSS, leupeptin and EBSS + leupeptin increased the secretion of endogenous tau by primary cortical neurons. (a) In normal conditions (CTL), one tau-positive band at 52 kDa was detected in the culture medium by an antibody revealing total tau (A0024). A similar pattern was observed when neurons were treated with leupeptin (L). When neurons were treated either with EBSS (E) or EBSS + leupeptin (E + L), three tau-positive bands at 47, 52 and 57 kDa were detected in the culture medium. In the cell lysate of control neurons, two tau-positive bands at 52 and 57 kDa were revealed by the anti-tau antibody A0024 (n = 6). (b) Quantification of total tau in culture medium by densitometry upon EBSS, leupeptin and E + L treatments (n = 6). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; *p

    Techniques Used: CTL Assay

    Significant increased secretion of dephosphorylated tau by EBSS and E + L treatments. (a) In the culture medium of control neurons (CTL) and leupeptin treated neurons (L), only one tau-positive band at 52 kDa was detected with the Tau-1 antibody. In the medium of EBSS (E) and E + L treated neurons, the bands at 47 and 52 kDa but not the one at 57 kDa were detected with this antibody (n = 6). (b) Quantification by densitometry of tau dephosphorylated at the Tau-1 antibody epitope in culture medium of control and E + L treated neurons (n = 11). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; **p
    Figure Legend Snippet: Significant increased secretion of dephosphorylated tau by EBSS and E + L treatments. (a) In the culture medium of control neurons (CTL) and leupeptin treated neurons (L), only one tau-positive band at 52 kDa was detected with the Tau-1 antibody. In the medium of EBSS (E) and E + L treated neurons, the bands at 47 and 52 kDa but not the one at 57 kDa were detected with this antibody (n = 6). (b) Quantification by densitometry of tau dephosphorylated at the Tau-1 antibody epitope in culture medium of control and E + L treated neurons (n = 11). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; **p

    Techniques Used: CTL Assay

    3) Product Images from "The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity"

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31364-y

    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P
    Figure Legend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Techniques Used: Activity Assay, Transfection, Staining

    4) Product Images from "Balance between autophagic pathways preserves retinal homeostasis"

    Article Title: Balance between autophagic pathways preserves retinal homeostasis

    Journal: Aging cell

    doi: 10.1111/acel.12072

    Impaired macroautophagy in the aged mouse retina . (A) Autophagic flux as determined by western blot of LC3 in whole retinal extracts from mice of different ages in the absence/presence of the lysosomal inhibitors ammonium chloride and leupeptin (N/L).
    Figure Legend Snippet: Impaired macroautophagy in the aged mouse retina . (A) Autophagic flux as determined by western blot of LC3 in whole retinal extracts from mice of different ages in the absence/presence of the lysosomal inhibitors ammonium chloride and leupeptin (N/L).

    Techniques Used: Western Blot, Mouse Assay

    5) Product Images from "The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity"

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31364-y

    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P
    Figure Legend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Techniques Used: Activity Assay, Transfection, Staining

    6) Product Images from "Chaperone-independent peripheral quality control of CFTR by RFFL E3 ligase"

    Article Title: Chaperone-independent peripheral quality control of CFTR by RFFL E3 ligase

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.02.001

    RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml leupeptin. Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .
    Figure Legend Snippet: RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml leupeptin. Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .

    Techniques Used: Fluorescence, Expressing, Transfection, Immunostaining, Labeling, Marker, Stable Transfection, Periodic Counter-current Chromatography

    7) Product Images from "HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers"

    Article Title: HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087675

    Analysis of the enzymatic activity of human HATL5. ( A ) Conditioned media samples from Pichia pastoris clones transfected with either the expression vector without protease insert (vector), with HATL5 serine protease domain cDNA (HATL5 #1 and #2) or matriptase serine protease domain cDNA were analyzed by western blotting using an anti-HATL5 antibody (left panel) or an anti-matriptase antibody (right panel). The positions of HATL5 (open arrow head) and matriptase serine protease domain (black arrow head) are indicated. ( B ) Samples from the conditioned media described in (A) were incubated at 37°C for 60 min with the synthetic chromogenic peptide Suc-Ala-Ala-Pro-Arg-pNA (100 µm) and the absorbance at 405 nm was recorded ( C ) 5 nM purified active recombinant HATL5 (white bars) or matriptase (black bars) serine protease domain was incubated at 37°C for 60 min with the synthetic chromogenic peptide MeOSuc-Glu-Val-Lys-Met-pNA (100 µm) in the absence or presence (inhibitor and substrate added concomitantly) of HAI-1 (60 nM), HAI-2 (40 nM), aprotinin (2 µm), leupeptin (20 µm), benzamidine (2 mM), or serpinA1 (60 nM). Enzyme activities for each enzyme are depicted relative to activity when no inhibitor was added.
    Figure Legend Snippet: Analysis of the enzymatic activity of human HATL5. ( A ) Conditioned media samples from Pichia pastoris clones transfected with either the expression vector without protease insert (vector), with HATL5 serine protease domain cDNA (HATL5 #1 and #2) or matriptase serine protease domain cDNA were analyzed by western blotting using an anti-HATL5 antibody (left panel) or an anti-matriptase antibody (right panel). The positions of HATL5 (open arrow head) and matriptase serine protease domain (black arrow head) are indicated. ( B ) Samples from the conditioned media described in (A) were incubated at 37°C for 60 min with the synthetic chromogenic peptide Suc-Ala-Ala-Pro-Arg-pNA (100 µm) and the absorbance at 405 nm was recorded ( C ) 5 nM purified active recombinant HATL5 (white bars) or matriptase (black bars) serine protease domain was incubated at 37°C for 60 min with the synthetic chromogenic peptide MeOSuc-Glu-Val-Lys-Met-pNA (100 µm) in the absence or presence (inhibitor and substrate added concomitantly) of HAI-1 (60 nM), HAI-2 (40 nM), aprotinin (2 µm), leupeptin (20 µm), benzamidine (2 mM), or serpinA1 (60 nM). Enzyme activities for each enzyme are depicted relative to activity when no inhibitor was added.

    Techniques Used: Activity Assay, Clone Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Incubation, Purification, Recombinant

    8) Product Images from "Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons"

    Article Title: Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons

    Journal: Scientific Reports

    doi: 10.1038/srep05715

    EBSS and E + L treatments induced AVs in primary cortical neurons. (a) LC3-II was analyzed by western blotting in the cell lysate prepared from EBSS (E), leupeptin (L) and EBSS + leupeptin (E + L) treated neurons. The LC3-II levels were compared to those of actin. For the analysis of LC3-II, 10 μg of cell lysate were loaded (n = 6). Values represent mean ± s.e.m.; *p
    Figure Legend Snippet: EBSS and E + L treatments induced AVs in primary cortical neurons. (a) LC3-II was analyzed by western blotting in the cell lysate prepared from EBSS (E), leupeptin (L) and EBSS + leupeptin (E + L) treated neurons. The LC3-II levels were compared to those of actin. For the analysis of LC3-II, 10 μg of cell lysate were loaded (n = 6). Values represent mean ± s.e.m.; *p

    Techniques Used: Western Blot

    EBSS, leupeptin and EBSS + leupeptin increased the secretion of endogenous tau by primary cortical neurons. (a) In normal conditions (CTL), one tau-positive band at 52 kDa was detected in the culture medium by an antibody revealing total tau (A0024). A similar pattern was observed when neurons were treated with leupeptin (L). When neurons were treated either with EBSS (E) or EBSS + leupeptin (E + L), three tau-positive bands at 47, 52 and 57 kDa were detected in the culture medium. In the cell lysate of control neurons, two tau-positive bands at 52 and 57 kDa were revealed by the anti-tau antibody A0024 (n = 6). (b) Quantification of total tau in culture medium by densitometry upon EBSS, leupeptin and E + L treatments (n = 6). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; *p
    Figure Legend Snippet: EBSS, leupeptin and EBSS + leupeptin increased the secretion of endogenous tau by primary cortical neurons. (a) In normal conditions (CTL), one tau-positive band at 52 kDa was detected in the culture medium by an antibody revealing total tau (A0024). A similar pattern was observed when neurons were treated with leupeptin (L). When neurons were treated either with EBSS (E) or EBSS + leupeptin (E + L), three tau-positive bands at 47, 52 and 57 kDa were detected in the culture medium. In the cell lysate of control neurons, two tau-positive bands at 52 and 57 kDa were revealed by the anti-tau antibody A0024 (n = 6). (b) Quantification of total tau in culture medium by densitometry upon EBSS, leupeptin and E + L treatments (n = 6). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; *p

    Techniques Used: CTL Assay

    Significant increased secretion of dephosphorylated tau by EBSS and E + L treatments. (a) In the culture medium of control neurons (CTL) and leupeptin treated neurons (L), only one tau-positive band at 52 kDa was detected with the Tau-1 antibody. In the medium of EBSS (E) and E + L treated neurons, the bands at 47 and 52 kDa but not the one at 57 kDa were detected with this antibody (n = 6). (b) Quantification by densitometry of tau dephosphorylated at the Tau-1 antibody epitope in culture medium of control and E + L treated neurons (n = 11). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; **p
    Figure Legend Snippet: Significant increased secretion of dephosphorylated tau by EBSS and E + L treatments. (a) In the culture medium of control neurons (CTL) and leupeptin treated neurons (L), only one tau-positive band at 52 kDa was detected with the Tau-1 antibody. In the medium of EBSS (E) and E + L treated neurons, the bands at 47 and 52 kDa but not the one at 57 kDa were detected with this antibody (n = 6). (b) Quantification by densitometry of tau dephosphorylated at the Tau-1 antibody epitope in culture medium of control and E + L treated neurons (n = 11). The intensities of the bands were expressed in arbitrary units. Values represent mean ± s.e.m.; **p

    Techniques Used: CTL Assay

    9) Product Images from "Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B, VesB *"

    Article Title: Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B, VesB *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525261

    Protease activity of purified VesB. A, VesB activity was measured with different concentrations of Boc-Gln-Ala-Arg-7-amino-4-AMC in 5 m m HEPES, pH 7.5, at 37 °C. B, purified VesB (0.08 μg/ml) was incubated with 50 μ m leupeptin,
    Figure Legend Snippet: Protease activity of purified VesB. A, VesB activity was measured with different concentrations of Boc-Gln-Ala-Arg-7-amino-4-AMC in 5 m m HEPES, pH 7.5, at 37 °C. B, purified VesB (0.08 μg/ml) was incubated with 50 μ m leupeptin,

    Techniques Used: Activity Assay, Purification, Incubation

    Related Articles

    Centrifugation:

    Article Title: Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization
    Article Snippet: .. Briefly, both untreated and stress-treated cells were collected; resuspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, and 0.6% Igepal) with protease inhibitors, 1 μm leupeptin, 1 μm E-64-d, and 1× HALT protease inhibitor mixture (Pierce); and incubated on ice for 20 min. After low-speed centrifugation for 10 min at 1,000 × g at 4°C, the supernatant was collected as cytoplasmic fraction lysate. .. The pellet was then washed and resuspended in buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM EDTA, and 1 mM EGTA supplemented with the same protease inhibitor mixture) on ice for 30 min.

    Magnetic Beads:

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    Protease Inhibitor:

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    Article Snippet: .. Equal numbers of sorted cells were rinsed in PBS, then lysed in buffer containing Tris HCl 25 mM, EDTA 1 mM, NaCl 130 mM, NP-40 1%; supplemented with protease inhibitor cocktail from Sigma Aldrich (St. Louis, MO) containing to final concentrations of 1.04 mM AEBSF, 800 μM Aprotinin, 40 μM Bestatin, 14 μM E-64, 20 μM Leupeptin, and15 μM Pepstatin A. Lysates were mixed with NuPAGE LDS loading buffer with 2.5% beta-mercaptoethanol, incubated at 95 °C for 2 minutes before loading and running on NuPAGE Novex 4–12% bis-tris gels (all products from Life Technologies, Grand Island, NY). .. Blots were transferred to Immobilon membrane (Millipore, Billerica, MA), prior to blotting with primary (IL-7Rα) followed by secondary (anti-rabbit-IgG-HRP) antibodies in TBS with 0.05% Tween-20 and 2% BSA.

    Article Title: Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization
    Article Snippet: .. Briefly, both untreated and stress-treated cells were collected; resuspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, and 0.6% Igepal) with protease inhibitors, 1 μm leupeptin, 1 μm E-64-d, and 1× HALT protease inhibitor mixture (Pierce); and incubated on ice for 20 min. After low-speed centrifugation for 10 min at 1,000 × g at 4°C, the supernatant was collected as cytoplasmic fraction lysate. .. The pellet was then washed and resuspended in buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM EDTA, and 1 mM EGTA supplemented with the same protease inhibitor mixture) on ice for 30 min.

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    Incubation:

    Article Title: Dissecting common ? chain cytokine family signaling in T cells using cell-to-cell variability analysis
    Article Snippet: .. Equal numbers of sorted cells were rinsed in PBS, then lysed in buffer containing Tris HCl 25 mM, EDTA 1 mM, NaCl 130 mM, NP-40 1%; supplemented with protease inhibitor cocktail from Sigma Aldrich (St. Louis, MO) containing to final concentrations of 1.04 mM AEBSF, 800 μM Aprotinin, 40 μM Bestatin, 14 μM E-64, 20 μM Leupeptin, and15 μM Pepstatin A. Lysates were mixed with NuPAGE LDS loading buffer with 2.5% beta-mercaptoethanol, incubated at 95 °C for 2 minutes before loading and running on NuPAGE Novex 4–12% bis-tris gels (all products from Life Technologies, Grand Island, NY). .. Blots were transferred to Immobilon membrane (Millipore, Billerica, MA), prior to blotting with primary (IL-7Rα) followed by secondary (anti-rabbit-IgG-HRP) antibodies in TBS with 0.05% Tween-20 and 2% BSA.

    Article Title: Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization
    Article Snippet: .. Briefly, both untreated and stress-treated cells were collected; resuspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, and 0.6% Igepal) with protease inhibitors, 1 μm leupeptin, 1 μm E-64-d, and 1× HALT protease inhibitor mixture (Pierce); and incubated on ice for 20 min. After low-speed centrifugation for 10 min at 1,000 × g at 4°C, the supernatant was collected as cytoplasmic fraction lysate. .. The pellet was then washed and resuspended in buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM EDTA, and 1 mM EGTA supplemented with the same protease inhibitor mixture) on ice for 30 min.

    Article Title: Myelin Proteolipid Protein Forms a Complex with Integrins and May Participate in Integrin Receptor Signaling in Oligodendrocytes
    Article Snippet: .. For immunoprecipitation reactions, lysates (1 mg/ml) were precleared in immunoprecipitation (IP) buffer: 0.15 m NaCl, 0.05 m Tris, 0.5 m m EDTA, 1% Triton X-100, 0.05% SDS, and 2% bovine serum albumin (BSA), pH 7.5, supplemented with protease inhibitors (20 μg/ml leupeptin, 100 μg/ml aprotinin, 2 m m PMSF, and 5 m m NEM) by incubation with appropriate species-specific IgG-conjugated magnetic beads (Dynabeads; Dynal, Lake Success, NY). .. After incubation at 4°C ON with gentle mixing, antibody–antigen complexes were captured with Dynabeads and washed.

    Mouse Assay:

    Article Title: Pten loss in Lgr5+ hair follicle stem cells promotes SCC development
    Article Snippet: .. Western blotting Freshly obtained skin samples from mice with hair removal were prepared in a lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2 mM CaCl2 and protease inhibitors (10 μg/mL leupeptin, 10 μg/mL aprotinin, 1.8 mg/mL iodoacetamide and 1 mmol/L phenylmethyl sulfonyl fluoride) and quantified with a BCA protein assay kit (Pierce). .. Equal amounts of total protein were subjected to electrophoresis on 12% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: anti-Pten (1:1000, 138G6, Cell Signaling Technology), anti-p-Akt (Ser473, 1:1500, GTX28932, GeneTex), anti-p-β-catenin antibody (Ser552, 1:1000, 5651S, Cell Signaling Technology), anti-p-Gsk-3β (Ser9) antibody (1:1000, 5558S, Cell Signaling Technology), and anti-p-β-catenin antibody (Ser675, 1:1000, 4176S, Cell Signaling Technology), anti-Akt antibody (1:1000, GTX121937, GeneTex), anti-β-catenin antibody (1:1000, 8480, Cell Signaling Technology), respectively, followed by a peroxidase-conjugated secondary antibody (KPL).

    Immunoprecipitation:

    Article Title: Myelin Proteolipid Protein Forms a Complex with Integrins and May Participate in Integrin Receptor Signaling in Oligodendrocytes
    Article Snippet: .. For immunoprecipitation reactions, lysates (1 mg/ml) were precleared in immunoprecipitation (IP) buffer: 0.15 m NaCl, 0.05 m Tris, 0.5 m m EDTA, 1% Triton X-100, 0.05% SDS, and 2% bovine serum albumin (BSA), pH 7.5, supplemented with protease inhibitors (20 μg/ml leupeptin, 100 μg/ml aprotinin, 2 m m PMSF, and 5 m m NEM) by incubation with appropriate species-specific IgG-conjugated magnetic beads (Dynabeads; Dynal, Lake Success, NY). .. After incubation at 4°C ON with gentle mixing, antibody–antigen complexes were captured with Dynabeads and washed.

    Protein Extraction:

    Article Title: Col1A1 Production and Apoptotic Resistance in TGF-?1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells
    Article Snippet: .. Western Blot Whole cell lysates were obtained with the M-PER Mammalian Protein Extraction reagent (Pierce) plus protease inhibitors (1 mmol/L phenylmethanesulfonylfluoride, 10 µg/mL leupeptin, and 2 µg/mL pepstatin). .. Antibodies to actin, caspase-3, E-cadherin (Sigma-Aldrich), N-cadherin (Cell Signaling), fibroblast-specific protein-1 (FSP-1), vimentin and Col1A1 (Abcam) were used.

    Expressing:

    Article Title: Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex
    Article Snippet: .. Reagents used for protein expression and purification include: 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, Fisher BioReagents 30827-99-7), phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich P7626), Tosyl-L-lysyl-chloromethane hydrochloride (TLCK, Sigma-Aldrich T7254), leupeptin (Thermo Scientific 78435), benzamidine (Sigma-Aldrich B6506), FLAG affinity resin (Sigma-Aldrich A2220), FLAG peptide (Sigma-Aldrich, F3290) and HIS-Select resin (Sigma-Aldrich P6611), and biotin (Sigma-Aldrich B4639). .. Cytoplasmic dynein and dynactin were purified from bovine brain as described previously , except that the preparation was scaled down to 1.5 brains (~300 g).

    BIA-KA:

    Article Title: Pten loss in Lgr5+ hair follicle stem cells promotes SCC development
    Article Snippet: .. Western blotting Freshly obtained skin samples from mice with hair removal were prepared in a lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2 mM CaCl2 and protease inhibitors (10 μg/mL leupeptin, 10 μg/mL aprotinin, 1.8 mg/mL iodoacetamide and 1 mmol/L phenylmethyl sulfonyl fluoride) and quantified with a BCA protein assay kit (Pierce). .. Equal amounts of total protein were subjected to electrophoresis on 12% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: anti-Pten (1:1000, 138G6, Cell Signaling Technology), anti-p-Akt (Ser473, 1:1500, GTX28932, GeneTex), anti-p-β-catenin antibody (Ser552, 1:1000, 5651S, Cell Signaling Technology), anti-p-Gsk-3β (Ser9) antibody (1:1000, 5558S, Cell Signaling Technology), and anti-p-β-catenin antibody (Ser675, 1:1000, 4176S, Cell Signaling Technology), anti-Akt antibody (1:1000, GTX121937, GeneTex), anti-β-catenin antibody (1:1000, 8480, Cell Signaling Technology), respectively, followed by a peroxidase-conjugated secondary antibody (KPL).

    Western Blot:

    Article Title: Pten loss in Lgr5+ hair follicle stem cells promotes SCC development
    Article Snippet: .. Western blotting Freshly obtained skin samples from mice with hair removal were prepared in a lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2 mM CaCl2 and protease inhibitors (10 μg/mL leupeptin, 10 μg/mL aprotinin, 1.8 mg/mL iodoacetamide and 1 mmol/L phenylmethyl sulfonyl fluoride) and quantified with a BCA protein assay kit (Pierce). .. Equal amounts of total protein were subjected to electrophoresis on 12% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: anti-Pten (1:1000, 138G6, Cell Signaling Technology), anti-p-Akt (Ser473, 1:1500, GTX28932, GeneTex), anti-p-β-catenin antibody (Ser552, 1:1000, 5651S, Cell Signaling Technology), anti-p-Gsk-3β (Ser9) antibody (1:1000, 5558S, Cell Signaling Technology), and anti-p-β-catenin antibody (Ser675, 1:1000, 4176S, Cell Signaling Technology), anti-Akt antibody (1:1000, GTX121937, GeneTex), anti-β-catenin antibody (1:1000, 8480, Cell Signaling Technology), respectively, followed by a peroxidase-conjugated secondary antibody (KPL).

    Article Title: Col1A1 Production and Apoptotic Resistance in TGF-?1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells
    Article Snippet: .. Western Blot Whole cell lysates were obtained with the M-PER Mammalian Protein Extraction reagent (Pierce) plus protease inhibitors (1 mmol/L phenylmethanesulfonylfluoride, 10 µg/mL leupeptin, and 2 µg/mL pepstatin). .. Antibodies to actin, caspase-3, E-cadherin (Sigma-Aldrich), N-cadherin (Cell Signaling), fibroblast-specific protein-1 (FSP-1), vimentin and Col1A1 (Abcam) were used.

    Lysis:

    Article Title: Pten loss in Lgr5+ hair follicle stem cells promotes SCC development
    Article Snippet: .. Western blotting Freshly obtained skin samples from mice with hair removal were prepared in a lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2 mM CaCl2 and protease inhibitors (10 μg/mL leupeptin, 10 μg/mL aprotinin, 1.8 mg/mL iodoacetamide and 1 mmol/L phenylmethyl sulfonyl fluoride) and quantified with a BCA protein assay kit (Pierce). .. Equal amounts of total protein were subjected to electrophoresis on 12% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: anti-Pten (1:1000, 138G6, Cell Signaling Technology), anti-p-Akt (Ser473, 1:1500, GTX28932, GeneTex), anti-p-β-catenin antibody (Ser552, 1:1000, 5651S, Cell Signaling Technology), anti-p-Gsk-3β (Ser9) antibody (1:1000, 5558S, Cell Signaling Technology), and anti-p-β-catenin antibody (Ser675, 1:1000, 4176S, Cell Signaling Technology), anti-Akt antibody (1:1000, GTX121937, GeneTex), anti-β-catenin antibody (1:1000, 8480, Cell Signaling Technology), respectively, followed by a peroxidase-conjugated secondary antibody (KPL).

    Article Title: trans-Resveratrol, an extract of red wine, inhibits human eosinophil activation and degranulation
    Article Snippet: .. Treated cells were harvested at indicated times and lysed in a lysis buffer containing 75 m M NaCl, 10 m M Tris-HCl, 1 m M EDTA, 0.5% NP40, 0.5% Triton-X100, 1 μg mL−1 leupeptin, 1 μg mL−1 aprotinin, 1 μ M phenylmethylsulphonyl fluoride, 1 m M Na3 VO4 and protease inhibitor cocktail (Pierce, Rockford, IL, USA). ..

    Article Title: Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines
    Article Snippet: .. Immunoblotting For immunoblotting of total and phosphorylated GSK3β and GLI1, cell lysate was extracted from A549 or H1915 cells with a lysis buffer (50 mM HEPES, 250 mM NaCl, 0.1% NP-40, 0.1 mM DTT) comprising a 1:00 dilution of protease inhibitor cocktail (Thermo Fisher Scientific Inc. Rockford, IL) containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, Pepstatin A), and a 1:00 dilution of phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc.) containing sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate. ..

    Purification:

    Article Title: Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex
    Article Snippet: .. Reagents used for protein expression and purification include: 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, Fisher BioReagents 30827-99-7), phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich P7626), Tosyl-L-lysyl-chloromethane hydrochloride (TLCK, Sigma-Aldrich T7254), leupeptin (Thermo Scientific 78435), benzamidine (Sigma-Aldrich B6506), FLAG affinity resin (Sigma-Aldrich A2220), FLAG peptide (Sigma-Aldrich, F3290) and HIS-Select resin (Sigma-Aldrich P6611), and biotin (Sigma-Aldrich B4639). .. Cytoplasmic dynein and dynactin were purified from bovine brain as described previously , except that the preparation was scaled down to 1.5 brains (~300 g).

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    Thermo Fisher leupeptin
    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; <t>Leupeptin,</t> 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P
    Leupeptin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Journal: Scientific Reports

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

    doi: 10.1038/s41598-018-31364-y

    Figure Lengend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Article Snippet: Briefly, cervical cancer cells were co-transfected with plasmids containing NaV 1.6 and GFP genes and seeded in the inserts at 1 × 105 cells density using culture medium with 5% FBS in absence or presence of 1-µM TTX, or in presence of protease inhibitors: GM6001, 25 µM (Millipore); E-64, 100 µM (Calbiochem; San Diego, CA); Leupeptin, 100 µM (Thermo Fisher Scientific), or the NHE-1 specific inhibitor 5-( N -ethyl- N -isopropyl) amiloride, EIPA, 1 µM (Sigma-Aldrich; St. Louis, MO).

    Techniques: Activity Assay, Transfection, Staining

    Impaired macroautophagy in the aged mouse retina . (A) Autophagic flux as determined by western blot of LC3 in whole retinal extracts from mice of different ages in the absence/presence of the lysosomal inhibitors ammonium chloride and leupeptin (N/L).

    Journal: Aging cell

    Article Title: Balance between autophagic pathways preserves retinal homeostasis

    doi: 10.1111/acel.12072

    Figure Lengend Snippet: Impaired macroautophagy in the aged mouse retina . (A) Autophagic flux as determined by western blot of LC3 in whole retinal extracts from mice of different ages in the absence/presence of the lysosomal inhibitors ammonium chloride and leupeptin (N/L).

    Article Snippet: Cells were treated with the combination of 100 μM leupeptin (Thermo Fisher Scientific, MA, USA) plus 20 mM ammonium chloride (Sigma) for 4h to inhibit lysosomal proteolysis.

    Techniques: Western Blot, Mouse Assay

    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Journal: Scientific Reports

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

    doi: 10.1038/s41598-018-31364-y

    Figure Lengend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Article Snippet: Briefly, cervical cancer cells were co-transfected with plasmids containing NaV 1.6 and GFP genes and seeded in the inserts at 1 × 105 cells density using culture medium with 5% FBS in absence or presence of 1-µM TTX, or in presence of protease inhibitors: GM6001, 25 µM (Millipore); E-64, 100 µM (Calbiochem; San Diego, CA); Leupeptin, 100 µM (Thermo Fisher Scientific), or the NHE-1 specific inhibitor 5-(N -ethyl-N -isopropyl) amiloride, EIPA, 1 µM (Sigma-Aldrich; St. Louis, MO).

    Techniques: Activity Assay, Transfection, Staining

    RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml leupeptin. Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .

    Journal: Developmental cell

    Article Title: Chaperone-independent peripheral quality control of CFTR by RFFL E3 ligase

    doi: 10.1016/j.devcel.2018.02.001

    Figure Lengend Snippet: RFFL ablation attenuates the endocytosis and lysosomal sorting of ΔF508-CFTR (A) Fluorescence micrographs of HeLa cells expressing RFFL-GFP or RFFLΔNT-GFP with mRFP-Rab5 or Lamp1-RFP. (B) Endocytosis rate of rΔF508-CFTR, TfR and CD4TccUbAllRΔG, (C) endocytic recycling of rΔF508-CFTR in CFBE upon 50 nM siRNA transfection. CHX chase experiment measuring stability of mature rΔF508-CFTR (D) and WT, T70 and P67L-CFTR-3HA (E). (F) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE immediately after 1 hour 37°C labeling (0 h) and 2 h chase. EEA1 was used as an early endosome marker. (G) Indirect immunostaining of internalized rΔF508-CFTR-3HA in CFBE after 4 h chase with 10 μg/ml leupeptin. Lysosome was labeled with Lysotracker Red. Co-localization of internalized rΔF508-CFTR-3HA with Lysotracker in shNT or shRFFL stably transfecting CFBE was quantified by Pearson’s correlation coefficient (PCC). (H) The mean vesicular pH of endocytic vesicles ( > 200 vesicles) containing rΔF508-CFTR or CD4Tl-λ L57C .

    Article Snippet: After solubilization in Tris-NP-40 solubilization buffer (20 mM Tris, 150 mM NaCl, 2 mM MgCl2 , 10% glycerol, 5 mM imidazole, and 0.1% NP-40 at pH 7.4) supplemented with 5 mM TCEP, 1 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml pepstatin A and 2.5 mM ATP, cell lysate was incubated with Neutravidin agarose (ThermoFisher) for 2 h at 4°C.

    Techniques: Fluorescence, Expressing, Transfection, Immunostaining, Labeling, Marker, Stable Transfection, Periodic Counter-current Chromatography