Structured Review

Boehringer Mannheim leupeptin
Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml <t>leupeptin</t> (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).
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1) Product Images from "Functional cleavage of the common cytokine receptor ? chain (?c) by calpain"

Article Title: Functional cleavage of the common cytokine receptor ? chain (?c) by calpain

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).
Figure Legend Snippet: Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

Techniques Used: In Vitro, Sequencing, Incubation, Protease Inhibitor, Immunoprecipitation, Staining

2) Product Images from "Intracellular re-routing of prion protein prevents propagation of PrPSc and delays onset of prion disease"

Article Title: Intracellular re-routing of prion protein prevents propagation of PrPSc and delays onset of prion disease

Journal: The EMBO Journal

doi: 10.1093/emboj/20.15.3957

Fig. 3. Suramin induces intracellular re-routing of PrP from post-ER compartments to acidic vesicles. ( A ) PrP is not detectable by surface FACS analysis in Suramin-treated cells. The left panel shows the surface FACS analysis of 3F4-N2a cells [the bold line is PrP reactivity without Suramin; the broken line is in the presence of Suramin (200 µg/ml for 24 h)]. The y -axis denotes the number of cells, the x -axis the fluorescence intensity (lg-scale). The polyclonal rabbit anti-PrP antibody A4 was used. The right panel shows the identical analysis with permeabilized cells (intracellular FACS). ( B ) No release of surface PrP by PIPLC in cells treated with Suramin. 3F4-N2a cells were incubated for 3 days with Suramin (200 µg/ml medium), treated with PIPLC, and the cellular lysate preparations analyzed in immunoblot (3F4). In non-Suramin-treated cells, PIPLC treatment results in the expected reduction of PrP in the cellular lysate fraction (lane 2); in the presence of Suramin, PIPLC treatment has no effect (lane 4). ( C ) Suramin induces intracellular retention of PrP in the Golgi/TGN and re-routing to acidic compartments. 3F4-N2a cells were mock treated (panel a) or incubated in the presence of Suramin (100µg/ml for 24 h) [panels (b) and (c)] and analyzed in indirect immunofluorescence experiments using confocal microscopy. (Panel a) PrP is excluded from the cell surface of Suramin-treated cells. Cell surface proteins of fixed and non-permeabilized 3F4-N2a cells were unspecifically surface-labeled with biotin; at the same time PrP present at the cell surface was specifically labeled with the antibody 3F4. The left panel shows surface biotinylated proteins (biotin), the middle panel cell surface PrP (3F4), and the right panel the overlay of both signals in yellowish color (merge). The upper right cell does not express detectable levels of 3F4-PrP, which indicates the specificity of the PrP antibody used. (Panel b) Suramin induces the retention of PrP in a Golgi/TGN compartment. 3F4-N2a cells were treated with Suramin, fixed, permeabilized and incubated with a polyclonal anti-mannosidase II antiserum to localize the medial to trans -Golgi compartments (left panel, manII). In parallel, PrP was analyzed with 3F4 (middle panel). In the right panel the overlay of both signals indicates co-localization (merge). (Panel c) PrP is re-routed to acidic compartments in Suramin-treated cells. Cells and treatment as in panel (b), with the exception that leupeptin was added. To stain for acidic endosomal/lysosomal compartments, an antibody against the acidotropic amine DAMP was used. The left panel shows the DAMP staining, the middle panel is PrP (3F4), and the right panel denotes the overlay of both signals (merge).
Figure Legend Snippet: Fig. 3. Suramin induces intracellular re-routing of PrP from post-ER compartments to acidic vesicles. ( A ) PrP is not detectable by surface FACS analysis in Suramin-treated cells. The left panel shows the surface FACS analysis of 3F4-N2a cells [the bold line is PrP reactivity without Suramin; the broken line is in the presence of Suramin (200 µg/ml for 24 h)]. The y -axis denotes the number of cells, the x -axis the fluorescence intensity (lg-scale). The polyclonal rabbit anti-PrP antibody A4 was used. The right panel shows the identical analysis with permeabilized cells (intracellular FACS). ( B ) No release of surface PrP by PIPLC in cells treated with Suramin. 3F4-N2a cells were incubated for 3 days with Suramin (200 µg/ml medium), treated with PIPLC, and the cellular lysate preparations analyzed in immunoblot (3F4). In non-Suramin-treated cells, PIPLC treatment results in the expected reduction of PrP in the cellular lysate fraction (lane 2); in the presence of Suramin, PIPLC treatment has no effect (lane 4). ( C ) Suramin induces intracellular retention of PrP in the Golgi/TGN and re-routing to acidic compartments. 3F4-N2a cells were mock treated (panel a) or incubated in the presence of Suramin (100µg/ml for 24 h) [panels (b) and (c)] and analyzed in indirect immunofluorescence experiments using confocal microscopy. (Panel a) PrP is excluded from the cell surface of Suramin-treated cells. Cell surface proteins of fixed and non-permeabilized 3F4-N2a cells were unspecifically surface-labeled with biotin; at the same time PrP present at the cell surface was specifically labeled with the antibody 3F4. The left panel shows surface biotinylated proteins (biotin), the middle panel cell surface PrP (3F4), and the right panel the overlay of both signals in yellowish color (merge). The upper right cell does not express detectable levels of 3F4-PrP, which indicates the specificity of the PrP antibody used. (Panel b) Suramin induces the retention of PrP in a Golgi/TGN compartment. 3F4-N2a cells were treated with Suramin, fixed, permeabilized and incubated with a polyclonal anti-mannosidase II antiserum to localize the medial to trans -Golgi compartments (left panel, manII). In parallel, PrP was analyzed with 3F4 (middle panel). In the right panel the overlay of both signals indicates co-localization (merge). (Panel c) PrP is re-routed to acidic compartments in Suramin-treated cells. Cells and treatment as in panel (b), with the exception that leupeptin was added. To stain for acidic endosomal/lysosomal compartments, an antibody against the acidotropic amine DAMP was used. The left panel shows the DAMP staining, the middle panel is PrP (3F4), and the right panel denotes the overlay of both signals (merge).

Techniques Used: FACS, Fluorescence, Incubation, Immunofluorescence, Confocal Microscopy, Labeling, Staining

3) Product Images from "Degradation of Mouse Invariant Chain: Roles of Cathepsins S and D and the Influence of Major Histocompatibility Complex Polymorphism "

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

Journal: The Journal of Experimental Medicine

doi:

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

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

4) Product Images from "Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM"

Article Title: Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM

Journal: The EMBO Journal

doi: 10.1093/emboj/19.5.882

Fig. 1. Ii is eliminated in ConB-treated cells without the intervention of cysteine proteases. ( A ) Mouse splenocytes were pulse-labeled for 30 min and chased for 60 and 240 min in the absence (control) or the presence of 1 mM leupeptin, 20 nM ConB or both drugs combined. MHC class II molecules were immunoprecipitated with mAb N22. Each immunoprecipitate was analyzed by reducing 12.5% SDS–PAGE without (NB) or after (B) boiling. The positions of immature (α o and β o ) and mature (α and β) I-A b ) and the Ii degradation intermediate IiP10 are indicated. SDS-stable αβ–Ii, αβ–peptide, αβ–IiP10 and (ConB-induced) αβc complexes are also indicated. The first lane (NRS) was loaded with an immunoprecipitate obtained with normal rabbit serum plus normal mouse serum from the lysate of control cells chased for 60 min. ( B ) N22 immunoprecipitates obtained from pulse-labeled cells, or cells chased for 240 min without (control) or with ConB or leupeptin as in (A), were denatured by boiling in SDS. One-tenth of the sample was set apart, and the remainder used for re-immunoprecipitation with anti-I-Aα and anti-I-Aβ rabbit sera, and sequentially with a rabbit serum against the N–terminal region of Ii. The N22 immunoprecipitate and each re-immunoprecipitated sample were loaded on a reducing 12.5% SDS–polyacrylamide gel. ( C ) Quantitation of the re-immunoprecipitations in (B). The amount of α, β and Ii in each of the pulse, control, leupeptin and ConB sets was quantitated in a phosphorimager. To correct for differences in the total amount of sample, the values in each set were normalized relative to β. The amount of each subunit relative to the amount present in the pulse-labeled sample was calculated.
Figure Legend Snippet: Fig. 1. Ii is eliminated in ConB-treated cells without the intervention of cysteine proteases. ( A ) Mouse splenocytes were pulse-labeled for 30 min and chased for 60 and 240 min in the absence (control) or the presence of 1 mM leupeptin, 20 nM ConB or both drugs combined. MHC class II molecules were immunoprecipitated with mAb N22. Each immunoprecipitate was analyzed by reducing 12.5% SDS–PAGE without (NB) or after (B) boiling. The positions of immature (α o and β o ) and mature (α and β) I-A b ) and the Ii degradation intermediate IiP10 are indicated. SDS-stable αβ–Ii, αβ–peptide, αβ–IiP10 and (ConB-induced) αβc complexes are also indicated. The first lane (NRS) was loaded with an immunoprecipitate obtained with normal rabbit serum plus normal mouse serum from the lysate of control cells chased for 60 min. ( B ) N22 immunoprecipitates obtained from pulse-labeled cells, or cells chased for 240 min without (control) or with ConB or leupeptin as in (A), were denatured by boiling in SDS. One-tenth of the sample was set apart, and the remainder used for re-immunoprecipitation with anti-I-Aα and anti-I-Aβ rabbit sera, and sequentially with a rabbit serum against the N–terminal region of Ii. The N22 immunoprecipitate and each re-immunoprecipitated sample were loaded on a reducing 12.5% SDS–polyacrylamide gel. ( C ) Quantitation of the re-immunoprecipitations in (B). The amount of α, β and Ii in each of the pulse, control, leupeptin and ConB sets was quantitated in a phosphorimager. To correct for differences in the total amount of sample, the values in each set were normalized relative to β. The amount of each subunit relative to the amount present in the pulse-labeled sample was calculated.

Techniques Used: Labeling, Immunoprecipitation, SDS Page, Quantitation Assay

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Western Blot:

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other:

Article Title: Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM
Article Snippet: Leupeptin was from Boehringer Mannheim (Indianapolis, IN) and ConB was obtained from Ajinimoto Co. (Kanagawa, Japan).

Protease Inhibitor:

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Lysis:

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    Boehringer Mannheim leupeptin
    Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml <t>leupeptin</t> (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).
    Leupeptin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Boehringer Mannheim reagents leupeptin
    Proteinase inhibitors reversibly inhibit the degradation of hemoglobin and the release of FPIX in Plasmodium falciparum . Cell suspensions were incubated for 3 h at 37°C in the absence or presence of 10 μM Ro 40-4388, 20 μM ALLM, 20 μM ALLN, or 20 μM <t>leupeptin.</t> Dark shaded bars show concentration of hemoglobin. Concentration of hemozoin is shown before inhibitor's washout (light shaded bars) and after washout with an additional 3-h incubation (unshaded bars). Percentage inhibition of hemoglobin digestion and hemozoin production are given above the bars. Data are means ± SD from five experiments.
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    88
    Boehringer Mannheim protease inhibitor leupeptin
    Fig. 7. Intracellular separation of gp40 and the arrested MHC class I complexes. B12 cells stably expressing gp40 were analyzed by confocal laser scanning microscopy. To inhibit lysosomal degradation, cells were incubated with 50 μg/ml <t>leupeptin</t> 12 h prior to double immunostaining. ( A ) The subcellular distribution of gp40 (green) was monitored with mAb gpM3D10, and MHC class I complexes (red) were detected with mAb SF1.1.1 (anti H2-K d ). ( B ) The staining pattern of gp40 (green) was compared with that of DAMP (red), a specific marker for acidic vesicles (endosomes/lysosomes). ( C ) Staining of MHC class I complexes (green) was compared with that of the ERGIC marker p58 (red).
    Protease Inhibitor Leupeptin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Functional cleavage of the common cytokine receptor ? chain (?c) by calpain

    doi:

    Figure Lengend Snippet: Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

    Article Snippet: Cells were lysed with Brij96 lysis buffer containing 5 mM EGTA and 2.5 mM EDTA and a protease inhibitor mix containing 10 μg/ml leupeptin (Boehringer Mannheim), 240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride (ICN), and 10 μg/ml aprotinin (ICN).

    Techniques: In Vitro, Sequencing, Incubation, Protease Inhibitor, Immunoprecipitation, Staining

    Fig. 3. Suramin induces intracellular re-routing of PrP from post-ER compartments to acidic vesicles. ( A ) PrP is not detectable by surface FACS analysis in Suramin-treated cells. The left panel shows the surface FACS analysis of 3F4-N2a cells [the bold line is PrP reactivity without Suramin; the broken line is in the presence of Suramin (200 µg/ml for 24 h)]. The y -axis denotes the number of cells, the x -axis the fluorescence intensity (lg-scale). The polyclonal rabbit anti-PrP antibody A4 was used. The right panel shows the identical analysis with permeabilized cells (intracellular FACS). ( B ) No release of surface PrP by PIPLC in cells treated with Suramin. 3F4-N2a cells were incubated for 3 days with Suramin (200 µg/ml medium), treated with PIPLC, and the cellular lysate preparations analyzed in immunoblot (3F4). In non-Suramin-treated cells, PIPLC treatment results in the expected reduction of PrP in the cellular lysate fraction (lane 2); in the presence of Suramin, PIPLC treatment has no effect (lane 4). ( C ) Suramin induces intracellular retention of PrP in the Golgi/TGN and re-routing to acidic compartments. 3F4-N2a cells were mock treated (panel a) or incubated in the presence of Suramin (100µg/ml for 24 h) [panels (b) and (c)] and analyzed in indirect immunofluorescence experiments using confocal microscopy. (Panel a) PrP is excluded from the cell surface of Suramin-treated cells. Cell surface proteins of fixed and non-permeabilized 3F4-N2a cells were unspecifically surface-labeled with biotin; at the same time PrP present at the cell surface was specifically labeled with the antibody 3F4. The left panel shows surface biotinylated proteins (biotin), the middle panel cell surface PrP (3F4), and the right panel the overlay of both signals in yellowish color (merge). The upper right cell does not express detectable levels of 3F4-PrP, which indicates the specificity of the PrP antibody used. (Panel b) Suramin induces the retention of PrP in a Golgi/TGN compartment. 3F4-N2a cells were treated with Suramin, fixed, permeabilized and incubated with a polyclonal anti-mannosidase II antiserum to localize the medial to trans -Golgi compartments (left panel, manII). In parallel, PrP was analyzed with 3F4 (middle panel). In the right panel the overlay of both signals indicates co-localization (merge). (Panel c) PrP is re-routed to acidic compartments in Suramin-treated cells. Cells and treatment as in panel (b), with the exception that leupeptin was added. To stain for acidic endosomal/lysosomal compartments, an antibody against the acidotropic amine DAMP was used. The left panel shows the DAMP staining, the middle panel is PrP (3F4), and the right panel denotes the overlay of both signals (merge).

    Journal: The EMBO Journal

    Article Title: Intracellular re-routing of prion protein prevents propagation of PrPSc and delays onset of prion disease

    doi: 10.1093/emboj/20.15.3957

    Figure Lengend Snippet: Fig. 3. Suramin induces intracellular re-routing of PrP from post-ER compartments to acidic vesicles. ( A ) PrP is not detectable by surface FACS analysis in Suramin-treated cells. The left panel shows the surface FACS analysis of 3F4-N2a cells [the bold line is PrP reactivity without Suramin; the broken line is in the presence of Suramin (200 µg/ml for 24 h)]. The y -axis denotes the number of cells, the x -axis the fluorescence intensity (lg-scale). The polyclonal rabbit anti-PrP antibody A4 was used. The right panel shows the identical analysis with permeabilized cells (intracellular FACS). ( B ) No release of surface PrP by PIPLC in cells treated with Suramin. 3F4-N2a cells were incubated for 3 days with Suramin (200 µg/ml medium), treated with PIPLC, and the cellular lysate preparations analyzed in immunoblot (3F4). In non-Suramin-treated cells, PIPLC treatment results in the expected reduction of PrP in the cellular lysate fraction (lane 2); in the presence of Suramin, PIPLC treatment has no effect (lane 4). ( C ) Suramin induces intracellular retention of PrP in the Golgi/TGN and re-routing to acidic compartments. 3F4-N2a cells were mock treated (panel a) or incubated in the presence of Suramin (100µg/ml for 24 h) [panels (b) and (c)] and analyzed in indirect immunofluorescence experiments using confocal microscopy. (Panel a) PrP is excluded from the cell surface of Suramin-treated cells. Cell surface proteins of fixed and non-permeabilized 3F4-N2a cells were unspecifically surface-labeled with biotin; at the same time PrP present at the cell surface was specifically labeled with the antibody 3F4. The left panel shows surface biotinylated proteins (biotin), the middle panel cell surface PrP (3F4), and the right panel the overlay of both signals in yellowish color (merge). The upper right cell does not express detectable levels of 3F4-PrP, which indicates the specificity of the PrP antibody used. (Panel b) Suramin induces the retention of PrP in a Golgi/TGN compartment. 3F4-N2a cells were treated with Suramin, fixed, permeabilized and incubated with a polyclonal anti-mannosidase II antiserum to localize the medial to trans -Golgi compartments (left panel, manII). In parallel, PrP was analyzed with 3F4 (middle panel). In the right panel the overlay of both signals indicates co-localization (merge). (Panel c) PrP is re-routed to acidic compartments in Suramin-treated cells. Cells and treatment as in panel (b), with the exception that leupeptin was added. To stain for acidic endosomal/lysosomal compartments, an antibody against the acidotropic amine DAMP was used. The left panel shows the DAMP staining, the middle panel is PrP (3F4), and the right panel denotes the overlay of both signals (merge).

    Article Snippet: Suramin and leupeptin (Boehringer Mannheim) were applied at 100 µg/ml (overnight and for 6 h, respectively).

    Techniques: FACS, Fluorescence, Incubation, Immunofluorescence, Confocal Microscopy, Labeling, Staining

    Proteinase inhibitors reversibly inhibit the degradation of hemoglobin and the release of FPIX in Plasmodium falciparum . Cell suspensions were incubated for 3 h at 37°C in the absence or presence of 10 μM Ro 40-4388, 20 μM ALLM, 20 μM ALLN, or 20 μM leupeptin. Dark shaded bars show concentration of hemoglobin. Concentration of hemozoin is shown before inhibitor's washout (light shaded bars) and after washout with an additional 3-h incubation (unshaded bars). Percentage inhibition of hemoglobin digestion and hemozoin production are given above the bars. Data are means ± SD from five experiments.

    Journal: The Journal of Cell Biology

    Article Title: Cellular Uptake of Chloroquine Is Dependent on Binding to Ferriprotoporphyrin IX and Is Independent of NHE Activity in Plasmodium falciparum

    doi:

    Figure Lengend Snippet: Proteinase inhibitors reversibly inhibit the degradation of hemoglobin and the release of FPIX in Plasmodium falciparum . Cell suspensions were incubated for 3 h at 37°C in the absence or presence of 10 μM Ro 40-4388, 20 μM ALLM, 20 μM ALLN, or 20 μM leupeptin. Dark shaded bars show concentration of hemoglobin. Concentration of hemozoin is shown before inhibitor's washout (light shaded bars) and after washout with an additional 3-h incubation (unshaded bars). Percentage inhibition of hemoglobin digestion and hemozoin production are given above the bars. Data are means ± SD from five experiments.

    Article Snippet: Reagents Leupeptin and trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E64) were obtained from Boehringer Mannheim .

    Techniques: Incubation, Concentration Assay, Inhibition

    The effect of various proteinase inhibitors on the steady-state uptake (CAR) and activity of CQ. (A) The following inhibitors are shown: E64 (closed square); leupeptin (open circle); Ro 61-9379 (closed triangle); Ro 61-7835 (open square); ALLN (closed inverted triangle); ALLM (open inverted triangle); and Ro 40-4388 (closed circle). (B) The reversible effect of proteinase inhibitors on the steady-state accumulation of CQ by CQS (HB3) parasites. Control data without inhibitor are shown by light shaded bars. Dark shaded bars show the effect before and after wash-off of 10 μM Ro 40-4388, 20 μM ALLM, and 20 μM ALLN. (C) The effect of the same concentrations of proteinase inhibitors (pre-wash) on the steady-state accumulation of CQ by CQR (K1) parasites in the absence (light shaded bars) or presence (dark shaded bars) of 10 μM verapamil. The effect of the inhibitors is reversible on washout (post-wash). Data are means ± SD from 10 experiments. (D) Antagonism of Ro 40-4388 and CQ against the CQS (HB3) parasite.

    Journal: The Journal of Cell Biology

    Article Title: Cellular Uptake of Chloroquine Is Dependent on Binding to Ferriprotoporphyrin IX and Is Independent of NHE Activity in Plasmodium falciparum

    doi:

    Figure Lengend Snippet: The effect of various proteinase inhibitors on the steady-state uptake (CAR) and activity of CQ. (A) The following inhibitors are shown: E64 (closed square); leupeptin (open circle); Ro 61-9379 (closed triangle); Ro 61-7835 (open square); ALLN (closed inverted triangle); ALLM (open inverted triangle); and Ro 40-4388 (closed circle). (B) The reversible effect of proteinase inhibitors on the steady-state accumulation of CQ by CQS (HB3) parasites. Control data without inhibitor are shown by light shaded bars. Dark shaded bars show the effect before and after wash-off of 10 μM Ro 40-4388, 20 μM ALLM, and 20 μM ALLN. (C) The effect of the same concentrations of proteinase inhibitors (pre-wash) on the steady-state accumulation of CQ by CQR (K1) parasites in the absence (light shaded bars) or presence (dark shaded bars) of 10 μM verapamil. The effect of the inhibitors is reversible on washout (post-wash). Data are means ± SD from 10 experiments. (D) Antagonism of Ro 40-4388 and CQ against the CQS (HB3) parasite.

    Article Snippet: Reagents Leupeptin and trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E64) were obtained from Boehringer Mannheim .

    Techniques: Activity Assay

    Fig. 7. Intracellular separation of gp40 and the arrested MHC class I complexes. B12 cells stably expressing gp40 were analyzed by confocal laser scanning microscopy. To inhibit lysosomal degradation, cells were incubated with 50 μg/ml leupeptin 12 h prior to double immunostaining. ( A ) The subcellular distribution of gp40 (green) was monitored with mAb gpM3D10, and MHC class I complexes (red) were detected with mAb SF1.1.1 (anti H2-K d ). ( B ) The staining pattern of gp40 (green) was compared with that of DAMP (red), a specific marker for acidic vesicles (endosomes/lysosomes). ( C ) Staining of MHC class I complexes (green) was compared with that of the ERGIC marker p58 (red).

    Journal: The EMBO Journal

    Article Title: The luminal part of the murine cytomegalovirus glycoprotein gp40 catalyzes the retention of MHC class I molecules

    doi: 10.1093/emboj/19.5.870

    Figure Lengend Snippet: Fig. 7. Intracellular separation of gp40 and the arrested MHC class I complexes. B12 cells stably expressing gp40 were analyzed by confocal laser scanning microscopy. To inhibit lysosomal degradation, cells were incubated with 50 μg/ml leupeptin 12 h prior to double immunostaining. ( A ) The subcellular distribution of gp40 (green) was monitored with mAb gpM3D10, and MHC class I complexes (red) were detected with mAb SF1.1.1 (anti H2-K d ). ( B ) The staining pattern of gp40 (green) was compared with that of DAMP (red), a specific marker for acidic vesicles (endosomes/lysosomes). ( C ) Staining of MHC class I complexes (green) was compared with that of the ERGIC marker p58 (red).

    Article Snippet: The protease inhibitor leupeptin (Boehringer, Mannheim) was used at a final concentration of 200 μM.

    Techniques: Stable Transfection, Expressing, Confocal Laser Scanning Microscopy, Incubation, Double Immunostaining, Staining, Marker

    Fig. 9. Transport dynamics of newly synthesized gp40 and MHC class I molecules. B12 cells stably expressing gp40 were treated for 10 h with 20 μg/ml cycloheximide to accumulate mRNA by inhibiting translation. After washing, cycloheximide was replaced by 5 μg/ml actinomycin D to inhibit further transcription and to allow translation of the accumulated mRNA. To avoid lysosomal degradation, 50 μg/ml leupeptin was added. Cells were fixed and permeabilized at the indicated times after addition of actinomycin D. ( A ) At 0.5 h after protein release, gp40 (green, mAb gpM3D10) co-localized with the MHC class I K d molecules (red, mAb SF1.1.1). ( B ) Two hours later, gp40 (green) co-localized with the medial – Golgi marker ManII (red). ( D) After 24 h, gp40 co-localized with DAMP, which stains acidic vesicles (endosomes/lysosomes). ( C and E ) During this time course, the retained MHC class I molecules (green) co-localized with the ERGIC marker p58 (red). Compared with the half-life of gp40, the time required for the transport of gp40 to the endosomes/lysosomes was increased by cycloheximide and actinomycin D treatment. This is probably due to delayed transport kinetics of the involved cellular compartments after protein release.

    Journal: The EMBO Journal

    Article Title: The luminal part of the murine cytomegalovirus glycoprotein gp40 catalyzes the retention of MHC class I molecules

    doi: 10.1093/emboj/19.5.870

    Figure Lengend Snippet: Fig. 9. Transport dynamics of newly synthesized gp40 and MHC class I molecules. B12 cells stably expressing gp40 were treated for 10 h with 20 μg/ml cycloheximide to accumulate mRNA by inhibiting translation. After washing, cycloheximide was replaced by 5 μg/ml actinomycin D to inhibit further transcription and to allow translation of the accumulated mRNA. To avoid lysosomal degradation, 50 μg/ml leupeptin was added. Cells were fixed and permeabilized at the indicated times after addition of actinomycin D. ( A ) At 0.5 h after protein release, gp40 (green, mAb gpM3D10) co-localized with the MHC class I K d molecules (red, mAb SF1.1.1). ( B ) Two hours later, gp40 (green) co-localized with the medial – Golgi marker ManII (red). ( D) After 24 h, gp40 co-localized with DAMP, which stains acidic vesicles (endosomes/lysosomes). ( C and E ) During this time course, the retained MHC class I molecules (green) co-localized with the ERGIC marker p58 (red). Compared with the half-life of gp40, the time required for the transport of gp40 to the endosomes/lysosomes was increased by cycloheximide and actinomycin D treatment. This is probably due to delayed transport kinetics of the involved cellular compartments after protein release.

    Article Snippet: The protease inhibitor leupeptin (Boehringer, Mannheim) was used at a final concentration of 200 μM.

    Techniques: Synthesized, Stable Transfection, Expressing, Marker