leupeptin  (Millipore)

 
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    Leupeptin
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    Structured Review

    Millipore leupeptin
    Leupeptin

    https://www.bioz.com/result/leupeptin/product/Millipore
    Average 99 stars, based on 1 article reviews
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    leupeptin - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy"

    Article Title: Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy

    Journal: Brain

    doi: 10.1093/brain/awy046

    Cellular stability of wild-type and human disease variants of Munc18-1 in HEK293 cells and neurons. ( A ) Immunochemistry of HEK293 cells infected with wild-type Munc18-1 (WT), C180Y, M443R, C522R and T574P constructs stained for Munc18-1, EGFP and Golgi marker (GM130). ( B ) Normalized Munc18-1 levels in HEK293 cells after viral infection with wild-type, C180Y, M443R, C522R and T574P constructs. The inset shows representative western blot of HEK293 cells after viral infection; n = 5, 5, 5, 2 and 2, respectively. ( C ) Western blot analysis of Munc18-1 protein levels 0, 6, 12, 24 and 30 h after block of protein synthesis with cycloheximide for HEK293 cells infected with wild-type, C180Y, M433R, C522R or T574P constructs. The infection with wild-type construct was used as a control for all performed western blot analysis. ( D ) Quantitative analysis of the Munc18-1 protein expression from western blots in HEK cells represented in C . ( E ) Western blot analysis of Munc18-1 protein levels 0, 12, 24 and 36 h after block of protein synthesis with cycloheximide for wild-type, C180Y, M433R, C522R or T574P constructs in Stxbp1 null neurons. The infection with wild-type construct was used as a control for all performed western blot analysis. ( F ) Quantitative analysis of the Munc18-1 protein expression from western blots in neurons represented in E . ( G–I ) Normalized Munc18-1 protein levels from three constructs expressed in HEK cells treated with MG132, Leupeptin or Pepstatin; n = 3, 2 and 2, respectively.
    Figure Legend Snippet: Cellular stability of wild-type and human disease variants of Munc18-1 in HEK293 cells and neurons. ( A ) Immunochemistry of HEK293 cells infected with wild-type Munc18-1 (WT), C180Y, M443R, C522R and T574P constructs stained for Munc18-1, EGFP and Golgi marker (GM130). ( B ) Normalized Munc18-1 levels in HEK293 cells after viral infection with wild-type, C180Y, M443R, C522R and T574P constructs. The inset shows representative western blot of HEK293 cells after viral infection; n = 5, 5, 5, 2 and 2, respectively. ( C ) Western blot analysis of Munc18-1 protein levels 0, 6, 12, 24 and 30 h after block of protein synthesis with cycloheximide for HEK293 cells infected with wild-type, C180Y, M433R, C522R or T574P constructs. The infection with wild-type construct was used as a control for all performed western blot analysis. ( D ) Quantitative analysis of the Munc18-1 protein expression from western blots in HEK cells represented in C . ( E ) Western blot analysis of Munc18-1 protein levels 0, 12, 24 and 36 h after block of protein synthesis with cycloheximide for wild-type, C180Y, M433R, C522R or T574P constructs in Stxbp1 null neurons. The infection with wild-type construct was used as a control for all performed western blot analysis. ( F ) Quantitative analysis of the Munc18-1 protein expression from western blots in neurons represented in E . ( G–I ) Normalized Munc18-1 protein levels from three constructs expressed in HEK cells treated with MG132, Leupeptin or Pepstatin; n = 3, 2 and 2, respectively.

    Techniques Used: Infection, Construct, Staining, Marker, Western Blot, Blocking Assay, Expressing

    2) Product Images from "Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy *"

    Article Title: Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.666354

    Autophagosome and lysosome fusion analysis and LC3 turnover assay in vitro . A , Western blot analysis of LC3 and the difference in the LC3-II levels between samples with and without bafilomycin A1 ( Baf A1 ), leupeptin ( LEU ), and chloroquine ( CQ ) was compared
    Figure Legend Snippet: Autophagosome and lysosome fusion analysis and LC3 turnover assay in vitro . A , Western blot analysis of LC3 and the difference in the LC3-II levels between samples with and without bafilomycin A1 ( Baf A1 ), leupeptin ( LEU ), and chloroquine ( CQ ) was compared

    Techniques Used: LC3 Turnover Assay, In Vitro, Western Blot

    3) Product Images from "Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes"

    Article Title: Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes

    Journal: The Journal of Cell Biology

    doi:

    Selective redistribution of newly synthesized MHC class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. ( A ) Leupeptin does not affect the density of compartments containing β-hexosaminidase and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin, cells were permitted to internalize Tfn coupled to HRP for 30 min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient) contained most of the β-hexosaminidase activity, while Tfn-HRP was mainly present in low density fractions. No difference was observed between leupeptin-treated and control cells. ( B ) Redistribution of newly synthesized MHC class II molecules into high density fractions in leupeptin-treated cells. After a pulse of [ 35 S]methionine and a 1-, 2-, or 4-h chase in the absence ( left ) or presence ( right ) of leupeptin, I-A b –expressing A20 cells were fractionated on Percoll gradients. The membranes in each of the gradient fractions were pelleted by centrifugation, lysed in Triton X-100, and then immunoprecipitated using mAbs to I-A b (Y3P). The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin ( bottom left ). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells and thus was not shown.
    Figure Legend Snippet: Selective redistribution of newly synthesized MHC class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. ( A ) Leupeptin does not affect the density of compartments containing β-hexosaminidase and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin, cells were permitted to internalize Tfn coupled to HRP for 30 min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient) contained most of the β-hexosaminidase activity, while Tfn-HRP was mainly present in low density fractions. No difference was observed between leupeptin-treated and control cells. ( B ) Redistribution of newly synthesized MHC class II molecules into high density fractions in leupeptin-treated cells. After a pulse of [ 35 S]methionine and a 1-, 2-, or 4-h chase in the absence ( left ) or presence ( right ) of leupeptin, I-A b –expressing A20 cells were fractionated on Percoll gradients. The membranes in each of the gradient fractions were pelleted by centrifugation, lysed in Triton X-100, and then immunoprecipitated using mAbs to I-A b (Y3P). The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin ( bottom left ). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells and thus was not shown.

    Techniques Used: Synthesized, Gradient Centrifugation, Incubation, Homogenization, Fractionation, Activity Assay, Expressing, Centrifugation, Immunoprecipitation, SDS Page

    Reversal of the leupeptin block results in MHC class II transport from lysosomes to the cell surface. ( A ) Reversibility of the effect of leupeptin on the intracellular retention of I-A b class II molecules. Cells were metabolically labeled for 20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total ( left ) or surface ( right ) I-A b molecules were immunoprecipitated sequentially. Upon removal of leupeptin, the total amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of peptide-loaded, SDS-stable compact dimers (“ C ”) increased. Ii-p10 completely disappeared over this time course. After a lag (see B ), compact dimers began to appear at the cell surface. ( B ) Kinetics of compact dimer formation and transport to the cell surface. Bands corresponding to total and surface compact dimers were quantified by phosphorimaging. The lag between formation of compact dimers and their subsequent appearance at the cell surface was 3–4 h.
    Figure Legend Snippet: Reversal of the leupeptin block results in MHC class II transport from lysosomes to the cell surface. ( A ) Reversibility of the effect of leupeptin on the intracellular retention of I-A b class II molecules. Cells were metabolically labeled for 20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total ( left ) or surface ( right ) I-A b molecules were immunoprecipitated sequentially. Upon removal of leupeptin, the total amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of peptide-loaded, SDS-stable compact dimers (“ C ”) increased. Ii-p10 completely disappeared over this time course. After a lag (see B ), compact dimers began to appear at the cell surface. ( B ) Kinetics of compact dimer formation and transport to the cell surface. Bands corresponding to total and surface compact dimers were quantified by phosphorimaging. The lag between formation of compact dimers and their subsequent appearance at the cell surface was 3–4 h.

    Techniques Used: Blocking Assay, Metabolic Labelling, Labeling, Incubation, Immunoprecipitation

    Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. ( A ) Quantification of MHC class II, Ii, and lgp-B in control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. ( B ) Immunogold localization of Ii chain, lgp-B, and I-A in leupeptin-treated cells. ( Upper panel ) Ultrathin cryosections were double immunogold labeled with the Ii chain antibody IN-1 (anti–Ii cytosolic domain; Ii CYT ) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. ( Lower panel ) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.
    Figure Legend Snippet: Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. ( A ) Quantification of MHC class II, Ii, and lgp-B in control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. ( B ) Immunogold localization of Ii chain, lgp-B, and I-A in leupeptin-treated cells. ( Upper panel ) Ultrathin cryosections were double immunogold labeled with the Ii chain antibody IN-1 (anti–Ii cytosolic domain; Ii CYT ) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. ( Lower panel ) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.

    Techniques Used: Labeling

    Intracellular retention of Ii-p10–associated class II molecules. Cells were pulsed for 20 min and chased for the indicated times (h) in the presence or absence of 2 mM leupeptin. At each time point, the cells were surface biotinylated before lysis. Total and cell surface biotinylated class II molecules were sequentially immunoprecipitated using the mAb Y3P and streptavidin-agarose. The samples were then analyzed by SDS-PAGE without boiling before electrophoresis. In untreated control cells ( top ), peptideloaded, 60-kD compact αβ dimers (“ C ”) began to appear both in total lysates and on the surface after 0.5 h of chase. A small amount of the p70 complex of Ii-p10–αβ dimers appeared transiently, beginning also at 0.5 h of chase. In leupeptin-treated cells ( bottom ), p70 began to accumulate in total lysates by 0.5 h, but little was recovered at the plasma membrane. Peptide-loaded compact dimers only began to appear after 2–4 h of chase both in lysates and at the cell surface.
    Figure Legend Snippet: Intracellular retention of Ii-p10–associated class II molecules. Cells were pulsed for 20 min and chased for the indicated times (h) in the presence or absence of 2 mM leupeptin. At each time point, the cells were surface biotinylated before lysis. Total and cell surface biotinylated class II molecules were sequentially immunoprecipitated using the mAb Y3P and streptavidin-agarose. The samples were then analyzed by SDS-PAGE without boiling before electrophoresis. In untreated control cells ( top ), peptideloaded, 60-kD compact αβ dimers (“ C ”) began to appear both in total lysates and on the surface after 0.5 h of chase. A small amount of the p70 complex of Ii-p10–αβ dimers appeared transiently, beginning also at 0.5 h of chase. In leupeptin-treated cells ( bottom ), p70 began to accumulate in total lysates by 0.5 h, but little was recovered at the plasma membrane. Peptide-loaded compact dimers only began to appear after 2–4 h of chase both in lysates and at the cell surface.

    Techniques Used: Lysis, Immunoprecipitation, SDS Page, Electrophoresis

    Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. ( A ) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-A b class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“ C ”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. ( Left ) Anode; ( right ) cathode. ( B ) I-A b – expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. ( C ) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.
    Figure Legend Snippet: Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. ( A ) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-A b class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“ C ”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. ( Left ) Anode; ( right ) cathode. ( B ) I-A b – expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. ( C ) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.

    Techniques Used: Fractionation, Flow Cytometry, Electrophoresis, Labeling, Centrifugation, Immunoprecipitation, SDS Page, Expressing, Marker

    Leupeptin treatment causes MHC class II molecules to accumulate in lgp-containing structures by immunofluorescence microscopy. Control or leupeptintreated (3 h, 2 mM leupeptin) I-A b –expressing A20 cells were fixed, permeabilized, and then stained for MHC class II (FITC, using mAb Y3P, two upper panels ) vs lgp-B (TRITC, using mAb GL2A7, lower two panels ). In control cells, the small amount of intracellular MHC class II was localized to structures that were generally negative for lgp-B. These probably represented CIIVs a nd early endosomes. Leupeptin treatment, however, induced extensive colocalization of class II and lgp-B.
    Figure Legend Snippet: Leupeptin treatment causes MHC class II molecules to accumulate in lgp-containing structures by immunofluorescence microscopy. Control or leupeptintreated (3 h, 2 mM leupeptin) I-A b –expressing A20 cells were fixed, permeabilized, and then stained for MHC class II (FITC, using mAb Y3P, two upper panels ) vs lgp-B (TRITC, using mAb GL2A7, lower two panels ). In control cells, the small amount of intracellular MHC class II was localized to structures that were generally negative for lgp-B. These probably represented CIIVs a nd early endosomes. Leupeptin treatment, however, induced extensive colocalization of class II and lgp-B.

    Techniques Used: Immunofluorescence, Microscopy, Expressing, Staining

    Immunogold localization of MHC class II molecules in leupeptin-treated cells. Ultrathin cryosections were immunogold labeled with the anti–class II mAb M5.114 and protein A–gold (PAG-10). ( A ) In control cells, MHC class II molecules are found on the plasma membrane, in intracellular compartments characterized by the presence of internal membranes. ( B ) In leupeptin-treated cells (18-h treatment), MHC class II molecules were detected on the plasma membrane and accumulate in electron-dense compartments displaying internal membranes. Bars, 120 nm.
    Figure Legend Snippet: Immunogold localization of MHC class II molecules in leupeptin-treated cells. Ultrathin cryosections were immunogold labeled with the anti–class II mAb M5.114 and protein A–gold (PAG-10). ( A ) In control cells, MHC class II molecules are found on the plasma membrane, in intracellular compartments characterized by the presence of internal membranes. ( B ) In leupeptin-treated cells (18-h treatment), MHC class II molecules were detected on the plasma membrane and accumulate in electron-dense compartments displaying internal membranes. Bars, 120 nm.

    Techniques Used: Labeling

    Leupeptin induces an accumulation of SDS- resistant I-A b Ii-p10 complexes. ( A ) Leupeptin induces the accumulation of 10-kD (Ii-p10) and 70-kD (p70) proteins that coprecipitate with I-A b . I-A b –expressing A20 cells were pulsed for 20 min with [ 35 S]methionine and chased at 37°C for the indicated times in the presence or absence of 2 mM leupeptin. After lysis, the I-A b molecules were immunoprecipitated using the Y3P mAb. The samples were not boiled before SDS-PAGE. Labeled class II molecules were not detected before 30 min of chase because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimers complexed with intact Ii chain. ( B ) p70 represents SDS-stable complexes containing class II α and β chains and a 10-kD protein. After a 20-min pulse and 4-h chase with or without 2 mM leupeptin (lanes Lp and C , respectively), class II molecules were immunoprecipitated using the Y3P mAb, and the samples were boiled ( B ) or not boiled ( NB ) before SDSPAGE. After boiling, p70 dissociated quantitatively into monomers corresponding to αβ and Ii-p10. ( C ) p70 represents SDS-stable I-A b αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with either anti–I-A b (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. ( D ) Kinetics of association between Ii-p10 and I-A b or I-A d . Pulse-chase experiments were performed as above using A20 cells expressing only I-A d or expressing both I-A d and I-A b . I-A d or I-A b –containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-A b persisted throughout the chase period, while Ii-p10–I-A d complexes appeared only transiently.
    Figure Legend Snippet: Leupeptin induces an accumulation of SDS- resistant I-A b Ii-p10 complexes. ( A ) Leupeptin induces the accumulation of 10-kD (Ii-p10) and 70-kD (p70) proteins that coprecipitate with I-A b . I-A b –expressing A20 cells were pulsed for 20 min with [ 35 S]methionine and chased at 37°C for the indicated times in the presence or absence of 2 mM leupeptin. After lysis, the I-A b molecules were immunoprecipitated using the Y3P mAb. The samples were not boiled before SDS-PAGE. Labeled class II molecules were not detected before 30 min of chase because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimers complexed with intact Ii chain. ( B ) p70 represents SDS-stable complexes containing class II α and β chains and a 10-kD protein. After a 20-min pulse and 4-h chase with or without 2 mM leupeptin (lanes Lp and C , respectively), class II molecules were immunoprecipitated using the Y3P mAb, and the samples were boiled ( B ) or not boiled ( NB ) before SDSPAGE. After boiling, p70 dissociated quantitatively into monomers corresponding to αβ and Ii-p10. ( C ) p70 represents SDS-stable I-A b αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with either anti–I-A b (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. ( D ) Kinetics of association between Ii-p10 and I-A b or I-A d . Pulse-chase experiments were performed as above using A20 cells expressing only I-A d or expressing both I-A d and I-A b . I-A d or I-A b –containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-A b persisted throughout the chase period, while Ii-p10–I-A d complexes appeared only transiently.

    Techniques Used: Expressing, Lysis, Immunoprecipitation, SDS Page, Labeling, Pulse Chase

    4) Product Images from "Beclin-1 regulates cigarette smoke–induced kidney injury in a murine model of chronic obstructive pulmonary disease"

    Article Title: Beclin-1 regulates cigarette smoke–induced kidney injury in a murine model of chronic obstructive pulmonary disease

    Journal: JCI Insight

    doi: 10.1172/jci.insight.99592

    Autophagy activity is induced in mouse kidneys after cigarette smoke exposure. ( A ) Scheme representing autophagic flux experiment in which leupeptin or bafilomycin A1 inhibits autophagosome degradation, leading to autophagosome accumulation. ( B ) Mice subjected to RA or CS for 2 months (left panel) or 6 months (right panel) were assayed for autophagic flux in vivo by injection with leupeptin or vehicle (PBS), followed by Western blotting for LC3B expression in kidney tissue after 2 and 6 months of exposure and Beclin-1 expression after 6 months of exposure. Dot plots represent quantitation of Western blots ( n = 3 per group, except for 6 months CS + leupeptin exposure, n = 2 per group). ( C ) HK-2 cells were exposed to CSE in the absence or presence of bafilomycin A1. Representative Western blot of Beclin-1 and LC3B expression to determine autophagic flux in vitro. Dot plots represent quantitation of 3 independent experiments. All data are mean ± SEM.* P
    Figure Legend Snippet: Autophagy activity is induced in mouse kidneys after cigarette smoke exposure. ( A ) Scheme representing autophagic flux experiment in which leupeptin or bafilomycin A1 inhibits autophagosome degradation, leading to autophagosome accumulation. ( B ) Mice subjected to RA or CS for 2 months (left panel) or 6 months (right panel) were assayed for autophagic flux in vivo by injection with leupeptin or vehicle (PBS), followed by Western blotting for LC3B expression in kidney tissue after 2 and 6 months of exposure and Beclin-1 expression after 6 months of exposure. Dot plots represent quantitation of Western blots ( n = 3 per group, except for 6 months CS + leupeptin exposure, n = 2 per group). ( C ) HK-2 cells were exposed to CSE in the absence or presence of bafilomycin A1. Representative Western blot of Beclin-1 and LC3B expression to determine autophagic flux in vitro. Dot plots represent quantitation of 3 independent experiments. All data are mean ± SEM.* P

    Techniques Used: Activity Assay, Mouse Assay, In Vivo, Injection, Western Blot, Expressing, Quantitation Assay, In Vitro

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    Article Snippet: Leupeptin and cycloheximide (Sigma) were used at 50 μg/ml and 40 μg/ml, respectively.

    Incubation:

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    Activation Assay:

    Article Title: Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria
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    Inhibition:

    Article Title: Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria
    Article Snippet: For glutamine supplementation experiments, cells were kept in 2 mM glutamine supplemented in 5 mM glucose- or 25 mM glucose-containing DMEM (as indicated in the figures) for 1 h. For the GDH assay, the cells were cultured in 5 mM glucose-containing DMEM. .. For AMPK activation, 0.5 mM AICAR (Sigma, catalog no. A9978) was used as a high dose, and 0.03 mM was used as a low dose for 1 h. For AMPK inhibition, compound C (dorsomorphin; Sigma, catalog no. A5499) was used at a 20 μM dose for 1 h. For increasing anaplerotic flux, 10 mM dimethyl α-ketoglutarate (DMKG; Sigma, catalog no. 349631) was added to cells for 1 h. For Arf1 inhibition, 10 μM brefeldin A was added in low-glucose conditions for 1 h. For inhibition of autophagy flux, the cells were kept in either low (5 mM)- or high (25 mM)-glucose medium with or without 100 μM leupeptin (Sigma, catalog no. L2884) for 12 h. For the qPCR and luciferase assays, the cells were kept in the indicated medium conditions for 6 h. ..

    Real-time Polymerase Chain Reaction:

    Article Title: Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria
    Article Snippet: For glutamine supplementation experiments, cells were kept in 2 mM glutamine supplemented in 5 mM glucose- or 25 mM glucose-containing DMEM (as indicated in the figures) for 1 h. For the GDH assay, the cells were cultured in 5 mM glucose-containing DMEM. .. For AMPK activation, 0.5 mM AICAR (Sigma, catalog no. A9978) was used as a high dose, and 0.03 mM was used as a low dose for 1 h. For AMPK inhibition, compound C (dorsomorphin; Sigma, catalog no. A5499) was used at a 20 μM dose for 1 h. For increasing anaplerotic flux, 10 mM dimethyl α-ketoglutarate (DMKG; Sigma, catalog no. 349631) was added to cells for 1 h. For Arf1 inhibition, 10 μM brefeldin A was added in low-glucose conditions for 1 h. For inhibition of autophagy flux, the cells were kept in either low (5 mM)- or high (25 mM)-glucose medium with or without 100 μM leupeptin (Sigma, catalog no. L2884) for 12 h. For the qPCR and luciferase assays, the cells were kept in the indicated medium conditions for 6 h. ..

    Luciferase:

    Article Title: Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria
    Article Snippet: For glutamine supplementation experiments, cells were kept in 2 mM glutamine supplemented in 5 mM glucose- or 25 mM glucose-containing DMEM (as indicated in the figures) for 1 h. For the GDH assay, the cells were cultured in 5 mM glucose-containing DMEM. .. For AMPK activation, 0.5 mM AICAR (Sigma, catalog no. A9978) was used as a high dose, and 0.03 mM was used as a low dose for 1 h. For AMPK inhibition, compound C (dorsomorphin; Sigma, catalog no. A5499) was used at a 20 μM dose for 1 h. For increasing anaplerotic flux, 10 mM dimethyl α-ketoglutarate (DMKG; Sigma, catalog no. 349631) was added to cells for 1 h. For Arf1 inhibition, 10 μM brefeldin A was added in low-glucose conditions for 1 h. For inhibition of autophagy flux, the cells were kept in either low (5 mM)- or high (25 mM)-glucose medium with or without 100 μM leupeptin (Sigma, catalog no. L2884) for 12 h. For the qPCR and luciferase assays, the cells were kept in the indicated medium conditions for 6 h. ..

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    • leupeptin  (Millipore)
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    Millipore leupeptin
    Phosphorylation of TAX1BP1 is required for localization to autolysosomes. (A) Immunofluorescence assays. TAX1BP1 KO HeLa cells were transfected with Flag-TAX1BP1 WT, 10A or 3SD (S254D, S593D and S666D) and 24 h later transfected with 2.5 μg/ml poly(I:C) for 6 h in the presence of 20 μM <t>leupeptin.</t> Scale bar, 10 μm. (B) Pearson’s correlation coefficient was calculated to measure co-localization between TAX1BP1 and LAMP1 in 8-12 cells randomly selected from each sample. Unpaired Student’s t -test, *** P
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    Phosphorylation of TAX1BP1 is required for localization to autolysosomes. (A) Immunofluorescence assays. TAX1BP1 KO HeLa cells were transfected with Flag-TAX1BP1 WT, 10A or 3SD (S254D, S593D and S666D) and 24 h later transfected with 2.5 μg/ml poly(I:C) for 6 h in the presence of 20 μM leupeptin. Scale bar, 10 μm. (B) Pearson’s correlation coefficient was calculated to measure co-localization between TAX1BP1 and LAMP1 in 8-12 cells randomly selected from each sample. Unpaired Student’s t -test, *** P

    Journal: bioRxiv

    Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by canonical and noncanonical IκB kinases promotes its lysosomal localization and clearance of MAVS aggregates

    doi: 10.1101/2021.01.07.425702

    Figure Lengend Snippet: Phosphorylation of TAX1BP1 is required for localization to autolysosomes. (A) Immunofluorescence assays. TAX1BP1 KO HeLa cells were transfected with Flag-TAX1BP1 WT, 10A or 3SD (S254D, S593D and S666D) and 24 h later transfected with 2.5 μg/ml poly(I:C) for 6 h in the presence of 20 μM leupeptin. Scale bar, 10 μm. (B) Pearson’s correlation coefficient was calculated to measure co-localization between TAX1BP1 and LAMP1 in 8-12 cells randomly selected from each sample. Unpaired Student’s t -test, *** P

    Article Snippet: ReagentsPoly(I:C), Bafilomycin A1, IKK inhibitor VII, and leupeptin were purchased from MilliporeSigma. λ-phosphatase was from New England Biolabs.

    Techniques: Immunofluorescence, Transfection

    Pharmacological inhibition of cathepsin B activity impairs AXL-dependent cell invasion in EAC cells. (A-F) Transwell invasion assay in the presence of leupeptin. (A) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (D) FLO-1 cells treated with vehicle or leupeptin (20 μM) for 48 hours. (B) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (F) FLO-1 cells per microscopic field after treatment with vehicle or leupeptin. Data are presented as median ± SD. (C) Trypan blue viability assay of SK-GT-4 cells or (F) FLO-1 cells following treatment with vehicle or leupeptin. (G-L) Transwell invasion assay in the presence of CA-074. (G) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (J) FLO-1 cells treated with vehicle or CA-074 (10 μM) for 48 hours. (H) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (K) FLO-1 cells per microscopic field after treatment with vehicle or CA-074. Data are presented as median ± SD. (I) Trypan blue-viability assay of SK-GT-4 cells or (L) FLO-1 cells following treatment with vehicle or CA-074.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: AXL Mediates Esophageal Adenocarcinoma Cell Invasion through Regulation of Extracellular Acidification and Lysosome Trafficking

    doi: 10.1016/j.neo.2018.08.005

    Figure Lengend Snippet: Pharmacological inhibition of cathepsin B activity impairs AXL-dependent cell invasion in EAC cells. (A-F) Transwell invasion assay in the presence of leupeptin. (A) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (D) FLO-1 cells treated with vehicle or leupeptin (20 μM) for 48 hours. (B) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (F) FLO-1 cells per microscopic field after treatment with vehicle or leupeptin. Data are presented as median ± SD. (C) Trypan blue viability assay of SK-GT-4 cells or (F) FLO-1 cells following treatment with vehicle or leupeptin. (G-L) Transwell invasion assay in the presence of CA-074. (G) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (J) FLO-1 cells treated with vehicle or CA-074 (10 μM) for 48 hours. (H) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (K) FLO-1 cells per microscopic field after treatment with vehicle or CA-074. Data are presented as median ± SD. (I) Trypan blue-viability assay of SK-GT-4 cells or (L) FLO-1 cells following treatment with vehicle or CA-074.

    Article Snippet: The cathepsin B inhibitors, leupeptin and CA-074, were obtained from Sigma-Aldrich and MedChem Express, respectively.

    Techniques: Inhibition, Activity Assay, Transwell Invasion Assay, Viability Assay

    1,25(OH) 2 D 3 induces proteasomal degradation of TXNIP in MCF-7 cells. a The reduction in TXNIP protein levels by 1,25(OH) 2 D 3 (100 nM) in MCF-7 cells is rescued by MG-132 (5 μM) or 2-deoxyglucose (10 mM), but not leupeptin (20 μM). The various molecules were added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated (66 h) MCF-7 cells, for an additional 6 h. b , c ITCH mRNA and protein expression is not markedly influenced by 1,25(OH) 2 D 3 . Relative expression was calculated using the ∆∆Ct method with vinculin as the housekeeping gene. Error bars ± SD; n > 3. d Overall protein ubiquitination in MCF-7 cells was not changed by 1,25(OH) 2 D 3 treatment. e Co-immunoprecipitation studies illustrate that the TXNIP-ITCH interaction is not altered by 1,25(OH) 2 D 3 treatment of MCF-7 cells. f Negative regulation of TXNIP protein expression by 1,25(OH) 2 D 3 is observed in MCF-7 cells with knocked-down AMPKα1 levels. g The non-calcemic 1,25(OH) 2 D 3 analogue, calcipotriol (100 nM; 72 h) induces similar effects on TXNIP expression as 1,25(OH) 2 D 3 . The cell permeable Ca 2+ chelator BAPTA-AM (20 μM) does not hamper 1 ,25(OH) 2 D 3 ’s effects on TXNIP expression. BAPTA-AM was added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated MCF-7 cells, 2 h prior to the end of the treatment period (72 h)

    Journal: Cancer & Metabolism

    Article Title: Activation of pro-survival metabolic networks by 1,25(OH)2D3 does not hamper the sensitivity of breast cancer cells to chemotherapeutics

    doi: 10.1186/s40170-018-0183-6

    Figure Lengend Snippet: 1,25(OH) 2 D 3 induces proteasomal degradation of TXNIP in MCF-7 cells. a The reduction in TXNIP protein levels by 1,25(OH) 2 D 3 (100 nM) in MCF-7 cells is rescued by MG-132 (5 μM) or 2-deoxyglucose (10 mM), but not leupeptin (20 μM). The various molecules were added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated (66 h) MCF-7 cells, for an additional 6 h. b , c ITCH mRNA and protein expression is not markedly influenced by 1,25(OH) 2 D 3 . Relative expression was calculated using the ∆∆Ct method with vinculin as the housekeeping gene. Error bars ± SD; n > 3. d Overall protein ubiquitination in MCF-7 cells was not changed by 1,25(OH) 2 D 3 treatment. e Co-immunoprecipitation studies illustrate that the TXNIP-ITCH interaction is not altered by 1,25(OH) 2 D 3 treatment of MCF-7 cells. f Negative regulation of TXNIP protein expression by 1,25(OH) 2 D 3 is observed in MCF-7 cells with knocked-down AMPKα1 levels. g The non-calcemic 1,25(OH) 2 D 3 analogue, calcipotriol (100 nM; 72 h) induces similar effects on TXNIP expression as 1,25(OH) 2 D 3 . The cell permeable Ca 2+ chelator BAPTA-AM (20 μM) does not hamper 1 ,25(OH) 2 D 3 ’s effects on TXNIP expression. BAPTA-AM was added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated MCF-7 cells, 2 h prior to the end of the treatment period (72 h)

    Article Snippet: Additional drugs used in the study were added to either fresh or conditioned medium for different time points (as described in the “ ” section) and included 10 mM 2-deoxyglucose (Fluka-Sigma-Aldrich, Germany), 5 μM MG-132 (Sigma-Aldrich, Germany), 20 μM leupeptin (Sigma-Aldrich, Germany), 100 nM calcipotriol (Cayman Chemical-Biomol GmbH, Germany), and 20 μM BAPTA-AM (Cayman Chemical-Biomol GmbH, Germany), as well as increasing concentrations of dehydroepiandrosterone (DHEA) (Cayman Chemical-Biomol GmbH, Germany), Na Oxamate (Cayman Chemical-Biomol GmbH, Germany), AZD-3965 (Cayman Chemical-Biomol GmbH, Germany), 5-fluorouracil (5-FU) (Fluka-Sigma-Aldrich, Germany), and CBR-5884 (Cayman Chemical-Biomol GmbH, Germany).

    Techniques: Expressing, Immunoprecipitation

    MCB-613 causes lysosome-mediated degradation on p53-R175H. a Effect of MCB-613 (2 h) on p53 mRNA levels in ALST and TYK-Nu cells. Values are normalized mean ± s.e.m. ( n = 3). b – d siRNA-mediated knockdown (48 h) of SRC-1 ( b ), SRC-2 ( c ), and SRC-3 ( d ) had no effect on p53-WT (ALST cells) and p53-R175H (TYK-Nu cells) levels. e shRNA-mediated knockdown of SRC3 (72 h) in TYK-Nu cells does not affect p53-R175H levels. f SRC-3 inhibitor SI2 (50 nM, 2 h) has no effect on p53-R175H levels in TYK-Nu cells. g , h MCB-613 caused a g decrease in the half-life of p53-R175H and h increase in the half-life of p53-WT. i Proteasome inhibitor MG132 does not inhibit MCB-613 induced turnover of p53-R175H. j Lysosome inhibitors (pepstatin A, Leupeptin, and E-64D) rescued MCB-613 induced turnover of p53-R175H

    Journal: Nature Communications

    Article Title: USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells

    doi: 10.1038/s41467-018-03599-w

    Figure Lengend Snippet: MCB-613 causes lysosome-mediated degradation on p53-R175H. a Effect of MCB-613 (2 h) on p53 mRNA levels in ALST and TYK-Nu cells. Values are normalized mean ± s.e.m. ( n = 3). b – d siRNA-mediated knockdown (48 h) of SRC-1 ( b ), SRC-2 ( c ), and SRC-3 ( d ) had no effect on p53-WT (ALST cells) and p53-R175H (TYK-Nu cells) levels. e shRNA-mediated knockdown of SRC3 (72 h) in TYK-Nu cells does not affect p53-R175H levels. f SRC-3 inhibitor SI2 (50 nM, 2 h) has no effect on p53-R175H levels in TYK-Nu cells. g , h MCB-613 caused a g decrease in the half-life of p53-R175H and h increase in the half-life of p53-WT. i Proteasome inhibitor MG132 does not inhibit MCB-613 induced turnover of p53-R175H. j Lysosome inhibitors (pepstatin A, Leupeptin, and E-64D) rescued MCB-613 induced turnover of p53-R175H

    Article Snippet: Reagents were obtained as indicated: cycloheximide (Sigma), MG132 (Sigma), E-64D (Sigma), pepstatin-A (Sigma), leupeptin (Sigma), lysotracker (Life Technologies), mitotracker (Life Technologies), MCB-613 (provided by Dr. David Lonard, BCM), PR-619 (Cayman chemicals), F6 or NSC632839 (Cayman chemicals), and IU-1 (Cayman chemicals).

    Techniques: shRNA