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  • 99
    Thermo Fisher leupeptin
    Leupeptin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore leupeptin
    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, <t>Leupeptin</t> or Pepstatin; n = 3, 2 and 2, respectively.
    Leupeptin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim leupeptin
    Role of Cat S on degradation of mouse Ii. ( A ) LHVS is a specific inhibitor of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated concentrations of LHVS or <t>leupeptin</t> (1 mM) followed by addition of Cbz–[ 125 I]–Tyr– Ala–CN 2 . The bands corresponding to Cat S and to the high and low molecular weight forms of Cat B are indicated. ( B ) A cys protease different from Cat S converts LIP22 into LIP10. H-2 d splenocytes were pulse labeled for 30 min and chased for 240 min without ( control ) or with 1 mM leupeptin or 3 nM LHVS. N22 immunoprecipitates were loaded without boiling in 12.5% SDS-PAGE. ( C ) Cat S cleaves Ii NH 2 terminally of CLIP. H-2 d splenocytes were pulse chased in the presence of LHVS and immunoprecipitated with N22. The precipitate was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C. After incubation, samples were boiled in 1% SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at the right of the figure indicates the position of CLIP-containing fragments devoid of the NH 2 -terminal region of Ii.
    Leupeptin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc leupeptin
    Role of Cat S on degradation of mouse Ii. ( A ) LHVS is a specific inhibitor of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated concentrations of LHVS or <t>leupeptin</t> (1 mM) followed by addition of Cbz–[ 125 I]–Tyr– Ala–CN 2 . The bands corresponding to Cat S and to the high and low molecular weight forms of Cat B are indicated. ( B ) A cys protease different from Cat S converts LIP22 into LIP10. H-2 d splenocytes were pulse labeled for 30 min and chased for 240 min without ( control ) or with 1 mM leupeptin or 3 nM LHVS. N22 immunoprecipitates were loaded without boiling in 12.5% SDS-PAGE. ( C ) Cat S cleaves Ii NH 2 terminally of CLIP. H-2 d splenocytes were pulse chased in the presence of LHVS and immunoprecipitated with N22. The precipitate was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C. After incubation, samples were boiled in 1% SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at the right of the figure indicates the position of CLIP-containing fragments devoid of the NH 2 -terminal region of Ii.
    Leupeptin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad leupeptin
    Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, <t>leupeptin</t> and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.
    Leupeptin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 633 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Beyotime leupeptin
    Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, <t>leupeptin</t> and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.
    Leupeptin, supplied by Beyotime, used in various techniques. Bioz Stars score: 96/100, based on 523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Peptide Institute leupeptin
    Number of autophagic vacuoles in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media containing lysosomal inhibitors (100 μM <t>leupeptin</t> and 1 μM pepstatin A). Representative fluorescence microscopy images of control (C-1) and LD (L-2 and M-1) fibroblasts incubated with anti-LC3 are shown. Bar: 10 μm. The histograms (C and D, for full and KH media, respectively) show the mean (value indicated above the corresponding bar) and standard deviation of the number of LC3 puncta, quantified in 50 cells from control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts in two different experiments (indicated in the histograms). Differences were found to be statistically significant at *P
    Leupeptin, supplied by Peptide Institute, used in various techniques. Bioz Stars score: 93/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology leupeptin
    Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with <t>leupeptin</t> (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P
    Leupeptin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Amresco leupeptin
    Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or <t>leupeptin</t> (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p
    Leupeptin, supplied by Amresco, used in various techniques. Bioz Stars score: 93/100, based on 207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific leupeptin
    Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and <t>leupeptin</t> (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as
    Leupeptin, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioShop leupeptin
    Rac1 promotes Rab5-dependent endocytosis of Crb. (A-E) Embryos were co-stained for Crb (red in the merged images) and Arm (green). Panels depict a surface view of the ventral ectoderm of the following embryos: Control (A: da-GAL4 embryo incubated in DMSO), Rac1 V12 (B: da-GAL4/UAS-Rac1 V12 embryo incubated in DMSO), Rac1 V12 <t>Leupeptin</t> (C: da-GAL4/UAS-Rac1 V12 embryo treated with the lysosomal proteolysis inhibitor leupeptin), Rac1 V12 Dynasore (D: da-GAL4/UAS-Rac1 V12 embryo treated with the dynamin inhibitor dynasore), Rac1 V12 Rab5 S43N ( da-GAL4/UAS-Rac1 V12 , UAS-Rab5 S43N ). Images are representative results of three independent experiments. Scale bar: 10 µm. C, arrows point to intracellular puncta; D,E, arrows indicate cell-cell borders. (F-H) Stage 16 Rab5 S43N -expressing embryos stained for 2A12 (F), Serp (G) or Verm (H). (I) 2A12 staining performed on an embryo expressing Rab5 S43N in the trachea and heterozygous for the null allele crb 11A22 ( btl-GAL4; UAS-Rab5 S43N / crb 11A22 ). Scale bar: 100 µm. (J) Histogram showing the quantification of the dorsal trunk length in Rab5 S43N ( btl-GAL4; UAS-Rab5 S43N ) and Rab5 S43N , crb /+ ( btl-GAL4; UAS-Rab5 S43N , crb 11A22 /+ ) embryos relative to dorsal trunk length of control animals ( btl-GAL4 ). n =15 embryos for each genotype, taken from at least three independent experiments. Bars represent mean±s.d. A two-tailed t -test was used to evaluate the statistical significance; ** P
    Leupeptin, supplied by BioShop, used in various techniques. Bioz Stars score: 93/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Applichem leupeptin
    Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), <t>leupeptin</t> (100 μM), CA-074 or
    Leupeptin, supplied by Applichem, used in various techniques. Bioz Stars score: 93/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant leupeptin
    Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), <t>leupeptin</t> (100 μM), CA-074 or
    Leupeptin, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bachem leupeptin
    Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), <t>leupeptin</t> (100 μM), CA-074 or
    Leupeptin, supplied by Bachem, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem leupeptin
    Lysosome proteases but not proteasome inhibitors decrease antigen cross-presentation induced by StII co-encapsulated with OVA into liposomes. Bone marrow-derived macrophages (BM-MΦs) from C57BL/6 mice were pre-incubated or not with 50 μM of lysosome protease inhibitors, <t>leupeptin,</t> and a cathepsin general inhibitor or with 1 μM of the proteasome inhibitor epoxomicin and stimulated with OVA co-encapsulated with StII into liposomes composed DPPC and Chol in 2:1 or 1:1 ratios [Lp/OVA/StII (2:1) or Lp/OVA/StII (1:1)] at final concentrations of 0.8 and 0.1 μg/ml of each protein, respectively. Cells stimulated with 25 μg/ml of OVA in soluble form and 10 nM of the SIINFEKL peptide were used as controls. Cells were washed and co-cultured with B3Z CD8 + T cells. β-galactosidase activity of the B3Z cells was determined by a colorimetric assay. Mean and SEM of OD 570nm of two independent experiments are shown. Statistical analysis was performed with the one-way ANOVA test with Dunnett post-test: * p
    Leupeptin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA leupeptin
    UL147A is an ER-resident protein which reduces surface MICA*008 but spares ER-resident MICA*008. (A) RKO MICA*008-HA cells transduced with an EV or with UL147A-FLAG were grown on glass slides, fixed and stained with an anti-protein disulfide isomerase (PDI) antibody (ER marker; red), an anti-FLAG-tag antibody (grey) and an anti-MICA antibody (green). Nuclei were stained with DAPI (blue). Images were captured by confocal microscopy. (B-C) RKO-MICA*008-HA cells expressing an EV (B) or N-terminally tagged UL147A (C) were left untreated (N), or incubated for 8 hours with the translation inhibitor cycloheximide (CHX, 50 μg/ml), in combination with one of two lysosomal inhibitors: <t>leupeptin</t> (LEU, 100 μg/ml) and concanamycin A (CCM A, 20 nM), or with one of two proteasomal inhibitors: epoxomicin (EPX, 8 μM) and bortezomib (BTZ, 8 μM). Each inhibitor was matched with an appropriate mock-treatment (DMSO or DDW). Following treatment, cells were lysed and blotted with anti-MICA. Anti-vinculin served as loading control. Representative of two independent experiments.
    Leupeptin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Biomedicals leupeptin
    UL147A is an ER-resident protein which reduces surface MICA*008 but spares ER-resident MICA*008. (A) RKO MICA*008-HA cells transduced with an EV or with UL147A-FLAG were grown on glass slides, fixed and stained with an anti-protein disulfide isomerase (PDI) antibody (ER marker; red), an anti-FLAG-tag antibody (grey) and an anti-MICA antibody (green). Nuclei were stained with DAPI (blue). Images were captured by confocal microscopy. (B-C) RKO-MICA*008-HA cells expressing an EV (B) or N-terminally tagged UL147A (C) were left untreated (N), or incubated for 8 hours with the translation inhibitor cycloheximide (CHX, 50 μg/ml), in combination with one of two lysosomal inhibitors: <t>leupeptin</t> (LEU, 100 μg/ml) and concanamycin A (CCM A, 20 nM), or with one of two proteasomal inhibitors: epoxomicin (EPX, 8 μM) and bortezomib (BTZ, 8 μM). Each inhibitor was matched with an appropriate mock-treatment (DMSO or DDW). Following treatment, cells were lysed and blotted with anti-MICA. Anti-vinculin served as loading control. Representative of two independent experiments.
    Leupeptin, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 92/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nacalai leupeptin
    UL147A is an ER-resident protein which reduces surface MICA*008 but spares ER-resident MICA*008. (A) RKO MICA*008-HA cells transduced with an EV or with UL147A-FLAG were grown on glass slides, fixed and stained with an anti-protein disulfide isomerase (PDI) antibody (ER marker; red), an anti-FLAG-tag antibody (grey) and an anti-MICA antibody (green). Nuclei were stained with DAPI (blue). Images were captured by confocal microscopy. (B-C) RKO-MICA*008-HA cells expressing an EV (B) or N-terminally tagged UL147A (C) were left untreated (N), or incubated for 8 hours with the translation inhibitor cycloheximide (CHX, 50 μg/ml), in combination with one of two lysosomal inhibitors: <t>leupeptin</t> (LEU, 100 μg/ml) and concanamycin A (CCM A, 20 nM), or with one of two proteasomal inhibitors: epoxomicin (EPX, 8 μM) and bortezomib (BTZ, 8 μM). Each inhibitor was matched with an appropriate mock-treatment (DMSO or DDW). Following treatment, cells were lysed and blotted with anti-MICA. Anti-vinculin served as loading control. Representative of two independent experiments.
    Leupeptin, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore leupeptin hydrochloride
    UL147A is an ER-resident protein which reduces surface MICA*008 but spares ER-resident MICA*008. (A) RKO MICA*008-HA cells transduced with an EV or with UL147A-FLAG were grown on glass slides, fixed and stained with an anti-protein disulfide isomerase (PDI) antibody (ER marker; red), an anti-FLAG-tag antibody (grey) and an anti-MICA antibody (green). Nuclei were stained with DAPI (blue). Images were captured by confocal microscopy. (B-C) RKO-MICA*008-HA cells expressing an EV (B) or N-terminally tagged UL147A (C) were left untreated (N), or incubated for 8 hours with the translation inhibitor cycloheximide (CHX, 50 μg/ml), in combination with one of two lysosomal inhibitors: <t>leupeptin</t> (LEU, 100 μg/ml) and concanamycin A (CCM A, 20 nM), or with one of two proteasomal inhibitors: epoxomicin (EPX, 8 μM) and bortezomib (BTZ, 8 μM). Each inhibitor was matched with an appropriate mock-treatment (DMSO or DDW). Following treatment, cells were lysed and blotted with anti-MICA. Anti-vinculin served as loading control. Representative of two independent experiments.
    Leupeptin Hydrochloride, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH leupeptin
    a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and <t>leupeptin</t> (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D
    Leupeptin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Brain

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

    doi: 10.1093/brain/awy046

    Figure Lengend 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.

    Article Snippet: For protease inhibition experiments, cells were incubated for 15 h with 100 µg/ml cycloheximide and 10 µM MG132 (BioConnect; sc-201270), 25 µM Leupeptin (Sigma; L2884) or 1 µM Pepstatin A (Sigma; P5318) or DMSO (1:500) after which cells were processed as described above.

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

    Role of Cat S on degradation of mouse Ii. ( A ) LHVS is a specific inhibitor of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated concentrations of LHVS or leupeptin (1 mM) followed by addition of Cbz–[ 125 I]–Tyr– Ala–CN 2 . The bands corresponding to Cat S and to the high and low molecular weight forms of Cat B are indicated. ( B ) A cys protease different from Cat S converts LIP22 into LIP10. H-2 d splenocytes were pulse labeled for 30 min and chased for 240 min without ( control ) or with 1 mM leupeptin or 3 nM LHVS. N22 immunoprecipitates were loaded without boiling in 12.5% SDS-PAGE. ( C ) Cat S cleaves Ii NH 2 terminally of CLIP. H-2 d splenocytes were pulse chased in the presence of LHVS and immunoprecipitated with N22. The precipitate was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C. After incubation, samples were boiled in 1% SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at the right of the figure indicates the position of CLIP-containing fragments devoid of the NH 2 -terminal region of Ii.

    Journal: The Journal of Experimental Medicine

    Article Title: Degradation of Mouse Invariant Chain: Roles of Cathepsins S and D and the Influence of Major Histocompatibility Complex Polymorphism

    doi:

    Figure Lengend Snippet: Role of Cat S on degradation of mouse Ii. ( A ) LHVS is a specific inhibitor of Cat S at the 1–10 nM range. Mouse splenocytes were incubated with the indicated concentrations of LHVS or leupeptin (1 mM) followed by addition of Cbz–[ 125 I]–Tyr– Ala–CN 2 . The bands corresponding to Cat S and to the high and low molecular weight forms of Cat B are indicated. ( B ) A cys protease different from Cat S converts LIP22 into LIP10. H-2 d splenocytes were pulse labeled for 30 min and chased for 240 min without ( control ) or with 1 mM leupeptin or 3 nM LHVS. N22 immunoprecipitates were loaded without boiling in 12.5% SDS-PAGE. ( C ) Cat S cleaves Ii NH 2 terminally of CLIP. H-2 d splenocytes were pulse chased in the presence of LHVS and immunoprecipitated with N22. The precipitate was resuspended in Cat S buffer and incubated with or without Cat S for 1 h at 37°C. After incubation, samples were boiled in 1% SDS, 1/5 loaded directly on gel, and the remainder diluted in lysis buffer and reimmunoprecipitated as in Fig. 1 B. The arrow at the right of the figure indicates the position of CLIP-containing fragments devoid of the NH 2 -terminal region of Ii.

    Article Snippet: Leupeptin was from Boehringer Mannheim (Indianapolis, IN).

    Techniques: Incubation, Molecular Weight, Labeling, SDS Page, Cross-linking Immunoprecipitation, Immunoprecipitation, Lysis

    Maturation of mouse MHC class II molecules in the absence or the presence of leupeptin. ( A ) Fresh H-2 d splenocytes were pulse labeled for 30 min and chased for the times indicated in the absence ( control ) or the presence of 1 mM leupeptin. MHC class II molecules were immunoprecipitated with mAb N22, and loaded on 12.5% reducing SDS-PAGE without ( NB ) or after ( B ) boiling. The position of the MHC class II α and β subunits, SDS-stable mature αβ heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated. Leupeptin-treated pulsed splenocytes showed the same pattern and intensity of bands as the control sample (data not shown). ( B ) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22 and LIP10. H-2 d splenocytes were pulse labeled for 30 min and chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH 2 -terminal or the CLIP region of Ii, or with mAb P4H5. Samples were loaded on 10% reducing SDS-PAGE with a fraction of the boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. ( C ) Structure of mouse Ii and the degradation intermediates that accumulate in mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and 119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references 6 , 7 . The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: Degradation of Mouse Invariant Chain: Roles of Cathepsins S and D and the Influence of Major Histocompatibility Complex Polymorphism

    doi:

    Figure Lengend Snippet: Maturation of mouse MHC class II molecules in the absence or the presence of leupeptin. ( A ) Fresh H-2 d splenocytes were pulse labeled for 30 min and chased for the times indicated in the absence ( control ) or the presence of 1 mM leupeptin. MHC class II molecules were immunoprecipitated with mAb N22, and loaded on 12.5% reducing SDS-PAGE without ( NB ) or after ( B ) boiling. The position of the MHC class II α and β subunits, SDS-stable mature αβ heterodimers, full-length Ii, and intermediate degradation products of Ii detected in the abscence (P10) or the presence of leupeptin (LIP25, LIP22, LIP18, and LIP10) are indicated. Leupeptin-treated pulsed splenocytes showed the same pattern and intensity of bands as the control sample (data not shown). ( B ) Reimmunoprecipitation of the leupeptin-induced polypeptides LIP22 and LIP10. H-2 d splenocytes were pulse labeled for 30 min and chased 240 min in the presence of 1 mM leupeptin. After immunoprecipitation with mAb N22, MHC class II molecules were fully denatured by boiling in 1% SDS and the released polypeptides immunoprecipitated in parallel with rabbit sera for the NH 2 -terminal or the CLIP region of Ii, or with mAb P4H5. Samples were loaded on 10% reducing SDS-PAGE with a fraction of the boiled N22 immunoprecipitate. The lower part of the panel corresponds to a longer exposure of the same gel shown in the upper half. ( C ) Structure of mouse Ii and the degradation intermediates that accumulate in mouse splenocytes treated with leupeptin (LIP22 and LIP10), as deduced from reimmunoprecipitations. The N-linked carbohydrates at positions 113 and 119, the region against which mAb P4H5 was raised, and the transmembrane, CLIP, and trimerization regions of Ii are indicated according to references 6 , 7 . The enzymes involved in each stage of degradation of Ii and the protease inhibitors that block those steps are indicated.

    Article Snippet: Leupeptin was from Boehringer Mannheim (Indianapolis, IN).

    Techniques: Labeling, Immunoprecipitation, SDS Page, Cross-linking Immunoprecipitation, Blocking Assay

    Maturation of I-A b proceeds normally in Cat D–deficient mice. Splenocytes from Cat D +/− ( top ) or Cat D −/− ( bottom ) littermates were pulse-chased in the absence or presence of leupeptin as indicated and I-A b molecules immunoprecipitated with mAb N22 and analyzed on 12.5% SDS-PAGE as in Fig. 1 B. The position of the I-A b αβ–LIP10 SDS-stable complex is indicated as αβl.

    Journal: The Journal of Experimental Medicine

    Article Title: Degradation of Mouse Invariant Chain: Roles of Cathepsins S and D and the Influence of Major Histocompatibility Complex Polymorphism

    doi:

    Figure Lengend Snippet: Maturation of I-A b proceeds normally in Cat D–deficient mice. Splenocytes from Cat D +/− ( top ) or Cat D −/− ( bottom ) littermates were pulse-chased in the absence or presence of leupeptin as indicated and I-A b molecules immunoprecipitated with mAb N22 and analyzed on 12.5% SDS-PAGE as in Fig. 1 B. The position of the I-A b αβ–LIP10 SDS-stable complex is indicated as αβl.

    Article Snippet: Leupeptin was from Boehringer Mannheim (Indianapolis, IN).

    Techniques: Mouse Assay, Immunoprecipitation, SDS Page

    The effect of cysteine protease inhibition on maturation of MHC class II molecules varies widely among allelic products. ( A ) Spleen cells of mice of the haplotypes indicated were pulse-labeled and chased for 4 h in the absence or presence of 1 mM leupeptin or 3 nM LHVS, and their I-A molecules immunoprecipitated with an anti-I-Aα rabbit serum ( 44 ). Immunoprecipitates were run on 12.5% SDS-PAGE without ( top half ) or after ( bottom half ) boiling. ( B ) Amount of I-A SDS-stable dimers generated in leupeptin-treated splenocytes of different haplotypes relative to their control counterparts. The amount of SDS-stable I-A d complexes in control cells was too small to perform a reliable comparison to the drug-treated samples. ( C ) Same as in B , for the LHVS-treated samples. ( D ) Cbz–[ 125 I]–Tyr–Ala–CN 2 labeling of H-2 d , H-2 b , and H-2 k splenocytes.

    Journal: The Journal of Experimental Medicine

    Article Title: Degradation of Mouse Invariant Chain: Roles of Cathepsins S and D and the Influence of Major Histocompatibility Complex Polymorphism

    doi:

    Figure Lengend Snippet: The effect of cysteine protease inhibition on maturation of MHC class II molecules varies widely among allelic products. ( A ) Spleen cells of mice of the haplotypes indicated were pulse-labeled and chased for 4 h in the absence or presence of 1 mM leupeptin or 3 nM LHVS, and their I-A molecules immunoprecipitated with an anti-I-Aα rabbit serum ( 44 ). Immunoprecipitates were run on 12.5% SDS-PAGE without ( top half ) or after ( bottom half ) boiling. ( B ) Amount of I-A SDS-stable dimers generated in leupeptin-treated splenocytes of different haplotypes relative to their control counterparts. The amount of SDS-stable I-A d complexes in control cells was too small to perform a reliable comparison to the drug-treated samples. ( C ) Same as in B , for the LHVS-treated samples. ( D ) Cbz–[ 125 I]–Tyr–Ala–CN 2 labeling of H-2 d , H-2 b , and H-2 k splenocytes.

    Article Snippet: Leupeptin was from Boehringer Mannheim (Indianapolis, IN).

    Techniques: Inhibition, Mouse Assay, Labeling, Immunoprecipitation, SDS Page, Generated

    Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, leupeptin and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.

    Journal: The international journal of biochemistry & cell biology

    Article Title: N-Terminal Truncation of Stat5a/b Circumvents PIAS3-Mediated Transcriptional Inhibition of Stat5 in Prostate Cancer Cells

    doi: 10.1016/j.biocel.2010.09.008

    Figure Lengend Snippet: Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, leupeptin and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.

    Article Snippet: Cells were lysed and fresh human prostate cancer specimens were homogenized in lysis buffer [10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulphonylfluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin], and the protein concentrations of the whole cell lysates were determined by the Bradford method (BioRad Laboratories Inc., Hercules, CA).

    Techniques: Electro Mobility Shift Assay, Generated, In Vitro, Lysis, Western Blot

    Number of autophagic vacuoles in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media containing lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A). Representative fluorescence microscopy images of control (C-1) and LD (L-2 and M-1) fibroblasts incubated with anti-LC3 are shown. Bar: 10 μm. The histograms (C and D, for full and KH media, respectively) show the mean (value indicated above the corresponding bar) and standard deviation of the number of LC3 puncta, quantified in 50 cells from control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts in two different experiments (indicated in the histograms). Differences were found to be statistically significant at *P

    Journal: The FEBS journal

    Article Title: Degradation of altered mitochondria by autophagy is impaired in Lafora disease

    doi: 10.1111/febs.14468

    Figure Lengend Snippet: Number of autophagic vacuoles in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media containing lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A). Representative fluorescence microscopy images of control (C-1) and LD (L-2 and M-1) fibroblasts incubated with anti-LC3 are shown. Bar: 10 μm. The histograms (C and D, for full and KH media, respectively) show the mean (value indicated above the corresponding bar) and standard deviation of the number of LC3 puncta, quantified in 50 cells from control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts in two different experiments (indicated in the histograms). Differences were found to be statistically significant at *P

    Article Snippet: Leupeptin was from Peptide Institute, Inc. and bovine serum albumin (BSA) from Roche Applied Science.

    Techniques: Incubation, Fluorescence, Microscopy, Standard Deviation

    Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls and laforin- and malin-deficient patients were stained with 100 nM MitoTracker Red for 30 min at 37 °C, washed and then treated or not with 10 μM CCCP in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2. Representative fluorescence microscopy images of control (C-1, A and B) and LD (L-2, C and D) fibroblasts, treated (B and D) or not (A and C) with CCCP. Two-dimensional scatter plots corresponding to red (mitochondria) and green (lysosomes) pixel intensities from B and D are shown on their rights. Bar: 10 μm. The histogram (E) shows the quantification (mean and standard deviation as percentage of the highest control value) of MitoTracker Red and LAMP2 colocalization after CCCP treatment in the different cells using MetaMorph software (CTR: mean values from C-1 and C2 cells; Laforin-: mean values from L-1 and L-2 cells; Malin-: M-1 cells). The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P

    Journal: The FEBS journal

    Article Title: Degradation of altered mitochondria by autophagy is impaired in Lafora disease

    doi: 10.1111/febs.14468

    Figure Lengend Snippet: Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls and laforin- and malin-deficient patients were stained with 100 nM MitoTracker Red for 30 min at 37 °C, washed and then treated or not with 10 μM CCCP in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2. Representative fluorescence microscopy images of control (C-1, A and B) and LD (L-2, C and D) fibroblasts, treated (B and D) or not (A and C) with CCCP. Two-dimensional scatter plots corresponding to red (mitochondria) and green (lysosomes) pixel intensities from B and D are shown on their rights. Bar: 10 μm. The histogram (E) shows the quantification (mean and standard deviation as percentage of the highest control value) of MitoTracker Red and LAMP2 colocalization after CCCP treatment in the different cells using MetaMorph software (CTR: mean values from C-1 and C2 cells; Laforin-: mean values from L-1 and L-2 cells; Malin-: M-1 cells). The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P

    Article Snippet: Leupeptin was from Peptide Institute, Inc. and bovine serum albumin (BSA) from Roche Applied Science.

    Techniques: Staining, Incubation, Fluorescence, Microscopy, Standard Deviation, Software

    LC3-II levels in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and patients with LD, laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of the CCCP treatment, fibroblasts were incubated in full or in KH media containing lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) were analysed by Western blot using anti-LC3 and anti-actin. Representative immunoblots for full (A) and KH (B) media are shown. The ratio of LC3-II to actin levels in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts was determined densitometrically and normalized in percentage to the highest control value (C and D, for full and KH media, respectively). All values are means and standard deviations of five independent experiments. Differences were found to be statistically significant at *P

    Journal: The FEBS journal

    Article Title: Degradation of altered mitochondria by autophagy is impaired in Lafora disease

    doi: 10.1111/febs.14468

    Figure Lengend Snippet: LC3-II levels in control and in LD fibroblasts in the presence or absence of CCCP Fibroblasts from controls (CTR: C-1 and C-2) and patients with LD, laforin- (Laforin-: L-1 and L-2) and malin-deficient (Malin-: M-1) were incubated for 18 h in full medium without (-CCCP) or with (+CCCP) 10 μM CCCP, as indicated. For the last 2 h of the CCCP treatment, fibroblasts were incubated in full or in KH media containing lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) were analysed by Western blot using anti-LC3 and anti-actin. Representative immunoblots for full (A) and KH (B) media are shown. The ratio of LC3-II to actin levels in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts was determined densitometrically and normalized in percentage to the highest control value (C and D, for full and KH media, respectively). All values are means and standard deviations of five independent experiments. Differences were found to be statistically significant at *P

    Article Snippet: Leupeptin was from Peptide Institute, Inc. and bovine serum albumin (BSA) from Roche Applied Science.

    Techniques: Incubation, Western Blot

    Effect of oligomycin plus antimycin A treatment on the LC3-II levels in control and in LD fibroblasts Control (C-1 and C-2), laforin-deficient (L-1 and L-2) and malin-deficient (M-1) fibroblasts were incubated in full medium without (-OA) or with (+OA) 10 μM oligomycin plus 1 μM antimycin A for 18 h. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media with lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) from the different samples were analysed by Western blot using anti-LC3 and anti-actin antibodies. Representative immunoblots are shown. The intensity of the bands was assessed by densitometry and the LC3-II values were normalized to those of actin in the same lanes (C and D, for full and KH media, respectively) in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts. In the histograms, the results are expressed as percentage of the respective highest control value. All values correspond to the means and standard deviations of five independent experiments. Differences were found to be statistically significant at **P

    Journal: The FEBS journal

    Article Title: Degradation of altered mitochondria by autophagy is impaired in Lafora disease

    doi: 10.1111/febs.14468

    Figure Lengend Snippet: Effect of oligomycin plus antimycin A treatment on the LC3-II levels in control and in LD fibroblasts Control (C-1 and C-2), laforin-deficient (L-1 and L-2) and malin-deficient (M-1) fibroblasts were incubated in full medium without (-OA) or with (+OA) 10 μM oligomycin plus 1 μM antimycin A for 18 h. For the last 2 h of treatment, fibroblasts were incubated in full (A) or in KH (B) media with lysosomal inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Cellular extracts (75 μg protein) from the different samples were analysed by Western blot using anti-LC3 and anti-actin antibodies. Representative immunoblots are shown. The intensity of the bands was assessed by densitometry and the LC3-II values were normalized to those of actin in the same lanes (C and D, for full and KH media, respectively) in control (CTR, red bars), laforin-deficient (Laforin-, green bars) and malin-deficient (Malin-, blue bars) fibroblasts. In the histograms, the results are expressed as percentage of the respective highest control value. All values correspond to the means and standard deviations of five independent experiments. Differences were found to be statistically significant at **P

    Article Snippet: Leupeptin was from Peptide Institute, Inc. and bovine serum albumin (BSA) from Roche Applied Science.

    Techniques: Incubation, Western Blot

    Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of oligomycin plus antimycin A Fibroblasts from controls (C-1 and C-2, A and B, respectively), and laforin- (L-1 and L-2, C and D, respectively) and malin-deficient (M-1, E) patients were treated with 10 μM oligomycin plus 1 μM antimycin A in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2 (green) and anti-TOM20 (red). Representative fluorescence microscopy images are shown. Bar: 10 μm. The histogram (F) shows the quantification (mean and standard deviation as percentage of the highest control value) of TOM20 and LAMP2 colocalization after oligomycin plus antimycin A treatment in the different cells using MetaMorph software. The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars, and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P

    Journal: The FEBS journal

    Article Title: Degradation of altered mitochondria by autophagy is impaired in Lafora disease

    doi: 10.1111/febs.14468

    Figure Lengend Snippet: Colocalization of mitochondrial and lysosomal markers in control and LD fibroblasts in the presence or absence of oligomycin plus antimycin A Fibroblasts from controls (C-1 and C-2, A and B, respectively), and laforin- (L-1 and L-2, C and D, respectively) and malin-deficient (M-1, E) patients were treated with 10 μM oligomycin plus 1 μM antimycin A in full medium for 18 h. Lysosomal inhibitors (100 μM leupeptin and 1 μM pepstatin A) were added for the last 2 h of the 18 h incubation period. Then, fibroblasts were immunostained with anti-LAMP2 (green) and anti-TOM20 (red). Representative fluorescence microscopy images are shown. Bar: 10 μm. The histogram (F) shows the quantification (mean and standard deviation as percentage of the highest control value) of TOM20 and LAMP2 colocalization after oligomycin plus antimycin A treatment in the different cells using MetaMorph software. The colocalization of mitochondria with lysosomes is reduced in LD fibroblasts (Laforin-, green bars, and Malin-, blue bars) compared to controls (CTR, red bars). Experiments were performed in duplicate, analysing 40 cells for each cell line. Differences were found to be statistically significant at ***P

    Article Snippet: Leupeptin was from Peptide Institute, Inc. and bovine serum albumin (BSA) from Roche Applied Science.

    Techniques: Incubation, Fluorescence, Microscopy, Standard Deviation, Software

    Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

    Journal: Nature immunology

    Article Title: Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells

    doi: 10.1038/s41590-017-0002-1

    Figure Lengend Snippet: Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

    Article Snippet: For immunofluorescence staining, HEK293T cells in which Atad3a was knocked down or not were seeded in the culture chamber and were treated with or without 10 μM leupeptin for 3 h, then fixed with 4% PFA, permeabilized with 0.1% Triton and stained with anti-Tom20 (sc-17764, Santa Cruz) and anti-Lamp2a (ab18528, Abcam).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Confocal Microscopy, Expressing, shRNA

    Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or leupeptin (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p

    Journal: Autophagy

    Article Title: Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

    doi: 10.1080/15548627.2016.1247143

    Figure Lengend Snippet: Blockage of autophagy flux is sufficient to induce cellular senescence. (A) NIH3T3 cells were treated with HCQ (3 μg/ml) or leupeptin (5 μg/ml) for 7 d. DCFH-DA fluorescence and SA-GLB1 staining was used to detect intracellular ROS (upper panel) and the senescent state (lower panel) of these cells. The percentages of SA-GLB1-positive cells were quantified. Scale bars: 50 μm. (B) Relative Il6 mRNA expression of the cells treated as described in (A) were quantified by qRT-PCR. (C) and (D) NIH3T3 cells transfected with control shRNA or Atg5 shRNA and positive cells were selected with 2 μg/ml puromycin for 7 d. Images of western blots for ATG5 (C) and SQSTM1 (D) are shown. (E) The cells described in (C) were cultured for another 5 d, and then treated with H 2 O 2 followed by a 3-d incubation. Images of SA-GLB1 staining and the percentages of SA-GLB1-positive cells are shown. Scale bars: 20 μm. (F) Relative Il6 mRNA level in cells cultured for 15 d were quantified by qRT-PCR. The data are presented as means ± SD from 3 independent experiments and **p

    Article Snippet: Leupeptin was from Amresco (J580).

    Techniques: Fluorescence, Staining, Expressing, Quantitative RT-PCR, Transfection, shRNA, Western Blot, Cell Culture, Incubation

    Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and leupeptin (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: Cells expressing CMA-mutant PLIN2 accumulate LD. ( a ) Immunoblot for indicated proteins of total cell lysates from untransfected cells (−) or cells transfected with wild-type (WT) or CMA-mutant (MT) PLIN2-GFP. o: overexpressed protein; e: endogenous protein. Representative blots of n=3 independent experiments. ( b ) Cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( c ) Cells expressing WT or MT PLIN2-GFP treated with OL with or without lysosomal inhibitors ammonium chloride and leupeptin (NL). Inset: Higher magnification of the boxed area. Graphs: average LD number or LD size. n=5 (LD number) and 3 (LD size) independent experiments with 40 cells per condition in each experiment. ( d ) Immunostaining for LAMP1 in cells treated as in (c). Top: Merged images. Bottom: Colocalized pixels in white. Insets: higher magnification. Graph: percentage colocalization of PLIN2-GFP with LAMP1. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Binding of WT and MT PLIN2-GFP from cellular extracts to GST-hsc70 immobilized on agarose beads. Representative blots of n=3 independent experiments. (f , g ) Immunostaining for hsc70 in OL-treated cells expressing WT or MT PLIN2-GFP. Colocalized pixels are in white. Insets: higher magnification. 3D-reconstruction shown in Supplementary Figure 4a . Graph: percentage colocalization of PLIN2-GFP with hsc70. n=5 independent experiments with 40 cells per condition in each experiment. ( h – j ) Live - cell imaging of cells cotransfected with WT or MT PLIN2-GFP and LAMP1-RFP and treated with OL. Sequential frames at 60s intervals are shown. Arrows: LD. Graphs: LD (i) kiss-run events/cell and (j) count/cell, tracking velocity, displacement and displacement rate. Small and large LD clusters defined as

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Expressing, Mutagenesis, Transfection, Immunostaining, Binding Assay, Live Cell Imaging, Cell Tracking Assay

    Failure to remove PLINs by CMA blocks macrolipophagy. ( a ) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Full fields shown in Supplementary Figure 5c . Graph: average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( b , c ) Cells stained as in (a) treated or not with rapamycin (Rapa). Insets: higher magnification. Graph: Average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( d ) Immunoblot for indicated proteins in homogenates (HOM) and lipid droplets (LD) isolated from livers for fed (F) and starved (S) rats. Representative blots of n=5 independent experiments. ( e , f ) Costaining for indicated ATG proteins and BODIPY493/503 in CTR and L2A(−) cells treated with OL. Insets: Higher magnification of the boxed areas with colocalized pixels in white. Graph: percentage colocalization with BODIPY. n=6 independent experiments with 40 cells per condition in each experiment. ( g ) Costaining for Rab7 in the same cells as in (e). Graph: percentage colocalization with BODIPY. n=4 independent experiments with 40 cells per condition in each experiment. ( h ) Immunoblot for indicated proteins of HOM and LD isolated from starved wild-type (+) or L2A knockout (−) mice livers. ( i ) Immunostaining for LC3 in cells expressing WT or MT PLIN2-GFP treated with OL. Inset: Higher magnification of the boxed area with colocalized pixels in white. 3D-reconstruction shown in Supplementary Figure 7c . Graph: percentage colocalization of LC3 with PLIN2-GFP. n=4 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ** P

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: Failure to remove PLINs by CMA blocks macrolipophagy. ( a ) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Full fields shown in Supplementary Figure 5c . Graph: average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( b , c ) Cells stained as in (a) treated or not with rapamycin (Rapa). Insets: higher magnification. Graph: Average LD number/cell. n=9 fields with 1350 cells per condition from 3 independent experiments. ( d ) Immunoblot for indicated proteins in homogenates (HOM) and lipid droplets (LD) isolated from livers for fed (F) and starved (S) rats. Representative blots of n=5 independent experiments. ( e , f ) Costaining for indicated ATG proteins and BODIPY493/503 in CTR and L2A(−) cells treated with OL. Insets: Higher magnification of the boxed areas with colocalized pixels in white. Graph: percentage colocalization with BODIPY. n=6 independent experiments with 40 cells per condition in each experiment. ( g ) Costaining for Rab7 in the same cells as in (e). Graph: percentage colocalization with BODIPY. n=4 independent experiments with 40 cells per condition in each experiment. ( h ) Immunoblot for indicated proteins of HOM and LD isolated from starved wild-type (+) or L2A knockout (−) mice livers. ( i ) Immunostaining for LC3 in cells expressing WT or MT PLIN2-GFP treated with OL. Inset: Higher magnification of the boxed area with colocalized pixels in white. 3D-reconstruction shown in Supplementary Figure 7c . Graph: percentage colocalization of LC3 with PLIN2-GFP. n=4 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ** P

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Staining, Isolation, Knock-Out, Mouse Assay, Immunostaining, Expressing

    PLIN2 and PLIN3 are CMA substrates. ( a ) Immunoblot for indicated proteins of total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: PLIN2 levels relative to untreated CTR cells. n=7 independent experiments. ( b ) Immunostaining for PLIN2 in CTR and L2A(−) cells treated as in (a). Graph: average puncta/cell. n=5 independent experiments with 40 cells per condition in each experiment. ( c ) Coimmunostaining for PLIN2 and LAMP1 in CTR and L2A(−) cells treated or not with OL followed by treatment with lysosomal inhibitors, ammonium chloride and leupeptin (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. ( d ) Graph: percentage colocalization of PLIN2 with LAMP1 from (c). n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins of homogenates (HOM), lysosomes with high (CMA+) and low (CMA−) activity isolated from fed or starved (Stv) rat livers. Representative blots from 5 independent experiments. ( f ) Immunoblot for indicated proteins of isolated lysosome-enriched fractions from OL-treated control and L2A(−) cells. ( g ) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). Graph: PLIN2 and PLIN3 levels in leupeptin-treated CMA+ lysosomes relative to untreated. Representative blots from 3 independent experiments (other two shown in Supplementary Figure 2h ). Values are mean ± SEM. Differences are significant for * P

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: PLIN2 and PLIN3 are CMA substrates. ( a ) Immunoblot for indicated proteins of total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: PLIN2 levels relative to untreated CTR cells. n=7 independent experiments. ( b ) Immunostaining for PLIN2 in CTR and L2A(−) cells treated as in (a). Graph: average puncta/cell. n=5 independent experiments with 40 cells per condition in each experiment. ( c ) Coimmunostaining for PLIN2 and LAMP1 in CTR and L2A(−) cells treated or not with OL followed by treatment with lysosomal inhibitors, ammonium chloride and leupeptin (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. ( d ) Graph: percentage colocalization of PLIN2 with LAMP1 from (c). n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins of homogenates (HOM), lysosomes with high (CMA+) and low (CMA−) activity isolated from fed or starved (Stv) rat livers. Representative blots from 5 independent experiments. ( f ) Immunoblot for indicated proteins of isolated lysosome-enriched fractions from OL-treated control and L2A(−) cells. ( g ) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). Graph: PLIN2 and PLIN3 levels in leupeptin-treated CMA+ lysosomes relative to untreated. Representative blots from 3 independent experiments (other two shown in Supplementary Figure 2h ). Values are mean ± SEM. Differences are significant for * P

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Incubation, Immunostaining, Activity Assay, Isolation

    Failure to remove PLINs by CMA blocks lipolysis. ( a ) Beta-oxidation rates in CTR and L2A(−) cells treated with OL followed by lipase inhibitor diethylumbelliferyl phosphate (Deup) and/or lysosomal inhibitors ammonium chloride and leupeptin (NL). n=3 independent experiments. ( b , c ) Costaining for DPH and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) after OL treatment. Colocalized pixels are in white. Right: Higher magnification of boxed area. Graph: percentage colocalization of DPH with ATGL. n=6 independent experiments with 40 cells per condition in each experiment. ( d ) Costaining for BODIPY493/503 and ATGL in CTR cells incubated in OL > S+ treated or not with atypical retinoic acid derivative (aRAd). Middle: Higher magnification area. Bottom: Colocalized pixels. Graph: percentage colocalization of BODIPY with ATGL. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: ATGL levels relative to untreated CTR cells. n=3 independent experiments. ( f , g ) Coimmunoprecipitation (IP) of ATGL in OL > S+ (f) or OL-treated (g) CTR (+) and L2A(−) cells. Cells in (f) are expressing G0S2-GFP. 1/10 input shown. Representative blots from n=3 (f) and 5 (g) independent experiments. ( h, i ) Immunoblot for indicated proteins of homogenates (HOM) and lipid droplets (LD) isolated from fed (F) or starved (S) rat livers (h), or starved wild-type (+) or L2A knockout (−) mice livers (i). Representative blots of n=4 (i; 3 additional blots shown in Supplementary Figure 5c ) and 7(h) independent experiments. ( j ) Immunostaining for ATGL in OL-treated cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graph: percentage colocalization of ATGL with PLIN2-GFP. n=5 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for * P

    Journal: Nature cell biology

    Article Title: Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    doi: 10.1038/ncb3166

    Figure Lengend Snippet: Failure to remove PLINs by CMA blocks lipolysis. ( a ) Beta-oxidation rates in CTR and L2A(−) cells treated with OL followed by lipase inhibitor diethylumbelliferyl phosphate (Deup) and/or lysosomal inhibitors ammonium chloride and leupeptin (NL). n=3 independent experiments. ( b , c ) Costaining for DPH and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) after OL treatment. Colocalized pixels are in white. Right: Higher magnification of boxed area. Graph: percentage colocalization of DPH with ATGL. n=6 independent experiments with 40 cells per condition in each experiment. ( d ) Costaining for BODIPY493/503 and ATGL in CTR cells incubated in OL > S+ treated or not with atypical retinoic acid derivative (aRAd). Middle: Higher magnification area. Bottom: Colocalized pixels. Graph: percentage colocalization of BODIPY with ATGL. n=5 independent experiments with 40 cells per condition in each experiment. ( e ) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented medium (OL > S+) or serum-deprived medium (OL > S−) after OL treatment. Graph: ATGL levels relative to untreated CTR cells. n=3 independent experiments. ( f , g ) Coimmunoprecipitation (IP) of ATGL in OL > S+ (f) or OL-treated (g) CTR (+) and L2A(−) cells. Cells in (f) are expressing G0S2-GFP. 1/10 input shown. Representative blots from n=3 (f) and 5 (g) independent experiments. ( h, i ) Immunoblot for indicated proteins of homogenates (HOM) and lipid droplets (LD) isolated from fed (F) or starved (S) rat livers (h), or starved wild-type (+) or L2A knockout (−) mice livers (i). Representative blots of n=4 (i; 3 additional blots shown in Supplementary Figure 5c ) and 7(h) independent experiments. ( j ) Immunostaining for ATGL in OL-treated cells expressing WT or MT PLIN2-GFP. Inset: Higher magnification of the boxed area. Graph: percentage colocalization of ATGL with PLIN2-GFP. n=5 independent experiments with 40 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for * P

    Article Snippet: Where indicated, OL was washed off with Hanks’ Balanced Salt Solution (Invitrogen) and replaced by DMEM or low-glucose DMEM with or without serum (OL > S+, OL > S+ Low Glc, OL > S-, respectively) for 16 h. Cells were treated with lysosomal inhibitors ammonium chloride (20 mM; American Bioanalyticals) and leupeptin (100 μM; Fisher Scientific), macroautophagy activator rapamycin (100 nM; Sigma-Aldrich), proteasomal inhibitors lactacystin (5 μM; Enzo Life Sciences) and MG132 (5 μM; Sigma-Aldrich), mitochondrial depolarizer FCCP (2 μM; Sigma-Aldrich) for 16 h unless indicated otherwise.

    Techniques: Incubation, Expressing, Isolation, Knock-Out, Mouse Assay, Immunostaining

    Rac1 promotes Rab5-dependent endocytosis of Crb. (A-E) Embryos were co-stained for Crb (red in the merged images) and Arm (green). Panels depict a surface view of the ventral ectoderm of the following embryos: Control (A: da-GAL4 embryo incubated in DMSO), Rac1 V12 (B: da-GAL4/UAS-Rac1 V12 embryo incubated in DMSO), Rac1 V12 Leupeptin (C: da-GAL4/UAS-Rac1 V12 embryo treated with the lysosomal proteolysis inhibitor leupeptin), Rac1 V12 Dynasore (D: da-GAL4/UAS-Rac1 V12 embryo treated with the dynamin inhibitor dynasore), Rac1 V12 Rab5 S43N ( da-GAL4/UAS-Rac1 V12 , UAS-Rab5 S43N ). Images are representative results of three independent experiments. Scale bar: 10 µm. C, arrows point to intracellular puncta; D,E, arrows indicate cell-cell borders. (F-H) Stage 16 Rab5 S43N -expressing embryos stained for 2A12 (F), Serp (G) or Verm (H). (I) 2A12 staining performed on an embryo expressing Rab5 S43N in the trachea and heterozygous for the null allele crb 11A22 ( btl-GAL4; UAS-Rab5 S43N / crb 11A22 ). Scale bar: 100 µm. (J) Histogram showing the quantification of the dorsal trunk length in Rab5 S43N ( btl-GAL4; UAS-Rab5 S43N ) and Rab5 S43N , crb /+ ( btl-GAL4; UAS-Rab5 S43N , crb 11A22 /+ ) embryos relative to dorsal trunk length of control animals ( btl-GAL4 ). n =15 embryos for each genotype, taken from at least three independent experiments. Bars represent mean±s.d. A two-tailed t -test was used to evaluate the statistical significance; ** P

    Journal: Biology Open

    Article Title: Rac1 controls epithelial tube length through the apical secretion and polarity pathways

    doi: 10.1242/bio.015727

    Figure Lengend Snippet: Rac1 promotes Rab5-dependent endocytosis of Crb. (A-E) Embryos were co-stained for Crb (red in the merged images) and Arm (green). Panels depict a surface view of the ventral ectoderm of the following embryos: Control (A: da-GAL4 embryo incubated in DMSO), Rac1 V12 (B: da-GAL4/UAS-Rac1 V12 embryo incubated in DMSO), Rac1 V12 Leupeptin (C: da-GAL4/UAS-Rac1 V12 embryo treated with the lysosomal proteolysis inhibitor leupeptin), Rac1 V12 Dynasore (D: da-GAL4/UAS-Rac1 V12 embryo treated with the dynamin inhibitor dynasore), Rac1 V12 Rab5 S43N ( da-GAL4/UAS-Rac1 V12 , UAS-Rab5 S43N ). Images are representative results of three independent experiments. Scale bar: 10 µm. C, arrows point to intracellular puncta; D,E, arrows indicate cell-cell borders. (F-H) Stage 16 Rab5 S43N -expressing embryos stained for 2A12 (F), Serp (G) or Verm (H). (I) 2A12 staining performed on an embryo expressing Rab5 S43N in the trachea and heterozygous for the null allele crb 11A22 ( btl-GAL4; UAS-Rab5 S43N / crb 11A22 ). Scale bar: 100 µm. (J) Histogram showing the quantification of the dorsal trunk length in Rab5 S43N ( btl-GAL4; UAS-Rab5 S43N ) and Rab5 S43N , crb /+ ( btl-GAL4; UAS-Rab5 S43N , crb 11A22 /+ ) embryos relative to dorsal trunk length of control animals ( btl-GAL4 ). n =15 embryos for each genotype, taken from at least three independent experiments. Bars represent mean±s.d. A two-tailed t -test was used to evaluate the statistical significance; ** P

    Article Snippet: Pharmacological treatment of embryos Dechorionated embryos were incubated in the dark at 25°C in a solution of 0.9% NaCl (under an octane phase) supplemented with DMSO (control), 0.8 mM Dynasore (30 min; Cedarlane) or 0.1 mg/ml leupeptin (3 h; Bioshop).

    Techniques: Staining, Incubation, Expressing, Two Tailed Test

    Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), leupeptin (100 μM), CA-074 or

    Journal: Journal of Virology

    Article Title: Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

    doi: 10.1128/JVI.00234-15

    Figure Lengend Snippet: Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), leupeptin (100 μM), CA-074 or

    Article Snippet: AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride], leupeptin, ZnCl2 , and ZnSO4 were from Applichem.

    Techniques: Infection

    Lysosome proteases but not proteasome inhibitors decrease antigen cross-presentation induced by StII co-encapsulated with OVA into liposomes. Bone marrow-derived macrophages (BM-MΦs) from C57BL/6 mice were pre-incubated or not with 50 μM of lysosome protease inhibitors, leupeptin, and a cathepsin general inhibitor or with 1 μM of the proteasome inhibitor epoxomicin and stimulated with OVA co-encapsulated with StII into liposomes composed DPPC and Chol in 2:1 or 1:1 ratios [Lp/OVA/StII (2:1) or Lp/OVA/StII (1:1)] at final concentrations of 0.8 and 0.1 μg/ml of each protein, respectively. Cells stimulated with 25 μg/ml of OVA in soluble form and 10 nM of the SIINFEKL peptide were used as controls. Cells were washed and co-cultured with B3Z CD8 + T cells. β-galactosidase activity of the B3Z cells was determined by a colorimetric assay. Mean and SEM of OD 570nm of two independent experiments are shown. Statistical analysis was performed with the one-way ANOVA test with Dunnett post-test: * p

    Journal: Frontiers in Immunology

    Article Title: The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes

    doi: 10.3389/fimmu.2018.02473

    Figure Lengend Snippet: Lysosome proteases but not proteasome inhibitors decrease antigen cross-presentation induced by StII co-encapsulated with OVA into liposomes. Bone marrow-derived macrophages (BM-MΦs) from C57BL/6 mice were pre-incubated or not with 50 μM of lysosome protease inhibitors, leupeptin, and a cathepsin general inhibitor or with 1 μM of the proteasome inhibitor epoxomicin and stimulated with OVA co-encapsulated with StII into liposomes composed DPPC and Chol in 2:1 or 1:1 ratios [Lp/OVA/StII (2:1) or Lp/OVA/StII (1:1)] at final concentrations of 0.8 and 0.1 μg/ml of each protein, respectively. Cells stimulated with 25 μg/ml of OVA in soluble form and 10 nM of the SIINFEKL peptide were used as controls. Cells were washed and co-cultured with B3Z CD8 + T cells. β-galactosidase activity of the B3Z cells was determined by a colorimetric assay. Mean and SEM of OD 570nm of two independent experiments are shown. Statistical analysis was performed with the one-way ANOVA test with Dunnett post-test: * p

    Article Snippet: Cathepsin general inhibitor (Z-FA-fmk) and leupeptin were purchased from ENZO life Science, Inc. (NY, United States) and Invitrogen, respectively.

    Techniques: Derivative Assay, Mouse Assay, Incubation, Cell Culture, Activity Assay, Colorimetric Assay

    UL147A is an ER-resident protein which reduces surface MICA*008 but spares ER-resident MICA*008. (A) RKO MICA*008-HA cells transduced with an EV or with UL147A-FLAG were grown on glass slides, fixed and stained with an anti-protein disulfide isomerase (PDI) antibody (ER marker; red), an anti-FLAG-tag antibody (grey) and an anti-MICA antibody (green). Nuclei were stained with DAPI (blue). Images were captured by confocal microscopy. (B-C) RKO-MICA*008-HA cells expressing an EV (B) or N-terminally tagged UL147A (C) were left untreated (N), or incubated for 8 hours with the translation inhibitor cycloheximide (CHX, 50 μg/ml), in combination with one of two lysosomal inhibitors: leupeptin (LEU, 100 μg/ml) and concanamycin A (CCM A, 20 nM), or with one of two proteasomal inhibitors: epoxomicin (EPX, 8 μM) and bortezomib (BTZ, 8 μM). Each inhibitor was matched with an appropriate mock-treatment (DMSO or DDW). Following treatment, cells were lysed and blotted with anti-MICA. Anti-vinculin served as loading control. Representative of two independent experiments.

    Journal: bioRxiv

    Article Title: The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

    doi: 10.1101/2020.07.17.208462

    Figure Lengend Snippet: UL147A is an ER-resident protein which reduces surface MICA*008 but spares ER-resident MICA*008. (A) RKO MICA*008-HA cells transduced with an EV or with UL147A-FLAG were grown on glass slides, fixed and stained with an anti-protein disulfide isomerase (PDI) antibody (ER marker; red), an anti-FLAG-tag antibody (grey) and an anti-MICA antibody (green). Nuclei were stained with DAPI (blue). Images were captured by confocal microscopy. (B-C) RKO-MICA*008-HA cells expressing an EV (B) or N-terminally tagged UL147A (C) were left untreated (N), or incubated for 8 hours with the translation inhibitor cycloheximide (CHX, 50 μg/ml), in combination with one of two lysosomal inhibitors: leupeptin (LEU, 100 μg/ml) and concanamycin A (CCM A, 20 nM), or with one of two proteasomal inhibitors: epoxomicin (EPX, 8 μM) and bortezomib (BTZ, 8 μM). Each inhibitor was matched with an appropriate mock-treatment (DMSO or DDW). Following treatment, cells were lysed and blotted with anti-MICA. Anti-vinculin served as loading control. Representative of two independent experiments.

    Article Snippet: Cycloheximide chase assay and proteasome and lysosome inhibitionCells were left untreated or incubated with 50 μg/ml cycloheximide (Sigma-Aldrich) for 8 h, in combination with mock treatment or with the following inhibitors: 100 μg/ml leupeptin (LEU; Merck Millipore), 20 nM concanamycin A (CCMA; Sigma-Aldrich), 8 μM epoxomicin (EPX; A2S), or 8 μM bortezomib (BTZ; LC Biolabs).

    Techniques: Transduction, Staining, Marker, FLAG-tag, Confocal Microscopy, Expressing, Incubation

    a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and leupeptin (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D

    Journal: Investigational New Drugs

    Article Title: Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

    doi: 10.1007/s10637-018-0569-x

    Figure Lengend Snippet: a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and leupeptin (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D

    Article Snippet: All fluorogenic substrates (Suc-Leu-Leu-Val-Tyr-amc, Ac-Arg-Leu-Arg-amc and Z-Leu-Leu-Glu-amc), the proteasome inhibitors Ac-APnLD-H and leupeptin, and all proteasome subunit-related antibodies (β1, β2, and β5) were purchased from Biomol (Plymouth Meeting, PA, U.S.A.).

    Techniques: Inhibition, Activity Assay