Structured Review

Peptide Institute pepstatin a
Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in  Rpe65 −/−  mice in retinal explants.  A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot.  B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads).  C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p
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Images

1) Product Images from "M-opsin protein degradation is inhibited by MG-132 in Rpe65−/− retinal explant culture"

Article Title: M-opsin protein degradation is inhibited by MG-132 in Rpe65−/− retinal explant culture

Journal: Molecular Vision

doi:

Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in  Rpe65 −/−  mice in retinal explants.  A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot.  B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads).  C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p
Figure Legend Snippet: Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in Rpe65 −/− mice in retinal explants. A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot. B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads). C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p

Techniques Used: Western Blot, Immunohistochemistry, Expressing, Protease Inhibitor, Mouse Assay, Staining, Labeling, Binding Assay

Expression of M-opsin protein in the retina after treatment with several types of protease inhibitors for 24 h in retinal explants. M-opsin protein levels were analyzed with western blotting after treatment with ( A ) pepstatin A, ( B ) E64d, and ( C ) MG-132.  D : Effect of MG-132 on M-opsin, S-opsin, GNAT2, GNAT1, and rhodopsin at the protein level analyzed with western blotting.  E : Quantitative analysis of M-opsin protein level.  F : Immunohistochemistry in MG-132-treated retinas for 24 h showed M-opsin (red). Cell nuclei were contrasted with DAPI (blue). M-opsin-positive cells increased after treatment with 2 μM or 10 μM MG-132 compared with vehicle in cone outer segments (arrows), ONL, and OPL (arrowheads) in the retina.  G : Densitometric analysis of immunofluorescence probed anti-M-opsin antibody in a 400-μm-wide section of retina. Histograms indicate mean±SD (n=6). *p
Figure Legend Snippet: Expression of M-opsin protein in the retina after treatment with several types of protease inhibitors for 24 h in retinal explants. M-opsin protein levels were analyzed with western blotting after treatment with ( A ) pepstatin A, ( B ) E64d, and ( C ) MG-132. D : Effect of MG-132 on M-opsin, S-opsin, GNAT2, GNAT1, and rhodopsin at the protein level analyzed with western blotting. E : Quantitative analysis of M-opsin protein level. F : Immunohistochemistry in MG-132-treated retinas for 24 h showed M-opsin (red). Cell nuclei were contrasted with DAPI (blue). M-opsin-positive cells increased after treatment with 2 μM or 10 μM MG-132 compared with vehicle in cone outer segments (arrows), ONL, and OPL (arrowheads) in the retina. G : Densitometric analysis of immunofluorescence probed anti-M-opsin antibody in a 400-μm-wide section of retina. Histograms indicate mean±SD (n=6). *p

Techniques Used: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

2) Product Images from "Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases"

Article Title: Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases

Journal: Biochemical Journal

doi: 10.1042/BJ20031575

Inhibition of nepenthesins by pepstatin and DAN ( A ) Pepstatin inhibition of nepenthesin I (•) at pH 3.0. Inhibition of porcine pepsin A (□) by pepstatin was also examined under the same conditions. ( B ) DAN inhibition of nepenthesin I in the presence (–•–) and absence (--•--) of cupric ions and of nepenthesin II in the presence (–○–) and absence (--○--) of cupric ions. Porcine pepsin A was treated with DAN in the same manner in the presence (–□–) and absence (--□--) of cupric ions.
Figure Legend Snippet: Inhibition of nepenthesins by pepstatin and DAN ( A ) Pepstatin inhibition of nepenthesin I (•) at pH 3.0. Inhibition of porcine pepsin A (□) by pepstatin was also examined under the same conditions. ( B ) DAN inhibition of nepenthesin I in the presence (–•–) and absence (--•--) of cupric ions and of nepenthesin II in the presence (–○–) and absence (--○--) of cupric ions. Porcine pepsin A was treated with DAN in the same manner in the presence (–□–) and absence (--□--) of cupric ions.

Techniques Used: Inhibition

3) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

4) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

5) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

6) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

7) Product Images from "Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4"

Article Title: Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4

Journal: Autophagy

doi: 10.1080/15548627.2017.1328348

Autophagy inhibition and enhanced unfolded protein responses (UPR) by AZM in association with STUB1 regulation and proteasome activation in LF. (A) WB using anti-LC3B and anti-ACTB of cell lysates from control (lane 1, 2) and AZM (lane 3, 4) treated LF in the presence or absence of protease inhibitors (E64d, pepstatin A). Protein samples were collected after 24-h treatment with AZM (10 μg/ml). In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p
Figure Legend Snippet: Autophagy inhibition and enhanced unfolded protein responses (UPR) by AZM in association with STUB1 regulation and proteasome activation in LF. (A) WB using anti-LC3B and anti-ACTB of cell lysates from control (lane 1, 2) and AZM (lane 3, 4) treated LF in the presence or absence of protease inhibitors (E64d, pepstatin A). Protein samples were collected after 24-h treatment with AZM (10 μg/ml). In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p

Techniques Used: Inhibition, Activation Assay, Western Blot, Expressing

Effect of AZM on TGFB-induced autophagy and TGFB-mediated UPR and proteasome activation in LF. (A) WB using anti-LC3 and anti-ACTB of cell lysates from control (lane 1, 2), TGFB1-treated (lane 3, 4, 7, 8) and AZM-treated (lane 5, 6, 7, 8) LF in the presence or absence of protease inhibitors (E64d, pepstatin A). Protein samples were collected after 24 h treatment with AZM (10 μg/ml) and TGFB1 (2 ng/ml). In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p
Figure Legend Snippet: Effect of AZM on TGFB-induced autophagy and TGFB-mediated UPR and proteasome activation in LF. (A) WB using anti-LC3 and anti-ACTB of cell lysates from control (lane 1, 2), TGFB1-treated (lane 3, 4, 7, 8) and AZM-treated (lane 5, 6, 7, 8) LF in the presence or absence of protease inhibitors (E64d, pepstatin A). Protein samples were collected after 24 h treatment with AZM (10 μg/ml) and TGFB1 (2 ng/ml). In the lower panel is the average ( ± SEM) taken from 3 independent experiments shown as relative expression. *p

Techniques Used: Activation Assay, Western Blot, Expressing

8) Product Images from "Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells"

Article Title: Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2013.1870

Cell growth inhibition and autophagy induction in MM cell lines after treatment with BZ. (A) U266, IM-9 and RPMI8226 cells were treated with BZ at various concentrations for 48 h. The number of viable cells was assessed by CellTiter Blue as described in Materials and methods. (B) U266 cells were cultured with or without BZ (10 nM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μ g/ml) and pepstatin A (10 μ g/ml) for 48 h. Cellular proteins were separated by 15% SDS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPDH mAb was performed as an internal control. The numbers indicate the ratio of LC3B-II/LC3B-I as determined by densitometry.
Figure Legend Snippet: Cell growth inhibition and autophagy induction in MM cell lines after treatment with BZ. (A) U266, IM-9 and RPMI8226 cells were treated with BZ at various concentrations for 48 h. The number of viable cells was assessed by CellTiter Blue as described in Materials and methods. (B) U266 cells were cultured with or without BZ (10 nM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μ g/ml) and pepstatin A (10 μ g/ml) for 48 h. Cellular proteins were separated by 15% SDS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPDH mAb was performed as an internal control. The numbers indicate the ratio of LC3B-II/LC3B-I as determined by densitometry.

Techniques Used: Inhibition, Cell Culture, SDS Page

Immunoblottings with anti-LC3B Ab and anti-p62 Ab after U266 cells were treated with various macrolide antibiotics. (A) U266 cells were treated with bafilomycin A 1 (10 nM), concanamycin A (10 nM), AZM (50 μ g/ml), EM (50 μ g/ml), or CAM (50 μ g/ml) for various lengths of time. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62 and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. (B) U266 cells were cultured with AZM (50 μ g/ml) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μ g/ml) and pepstatin A (10 μ g/ml) for 24 h. Cellular proteins were separated by SDS-PAGE and immunoblotted as described above. The numbers indicate the ratios of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.
Figure Legend Snippet: Immunoblottings with anti-LC3B Ab and anti-p62 Ab after U266 cells were treated with various macrolide antibiotics. (A) U266 cells were treated with bafilomycin A 1 (10 nM), concanamycin A (10 nM), AZM (50 μ g/ml), EM (50 μ g/ml), or CAM (50 μ g/ml) for various lengths of time. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62 and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. (B) U266 cells were cultured with AZM (50 μ g/ml) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μ g/ml) and pepstatin A (10 μ g/ml) for 24 h. Cellular proteins were separated by SDS-PAGE and immunoblotted as described above. The numbers indicate the ratios of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.

Techniques Used: Chick Chorioallantoic Membrane Assay, SDS Page, Cell Culture

9) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

10) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

11) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

12) Product Images from "Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines"

Article Title: Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines

Journal: International Journal of Oncology

doi: 10.3892/ijo.2015.3237

Autophagy induction after treatment with GEF. (A) PANC-1 cells and BxPC-3 cells were treated with GEF (10 and 25 μM) for 16 and 24 h. Cellular proteins were separated by 15% SDS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPDH mAb was performed as an internal control. Numbers indicating the expression ratios of LC3B-II/GAPDH were determined using densitometry. (B) PANC-1 cells and BxPC-3 cells were cultured with or without GEF (25 μM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml), and pepstatin A (10 μg/ml) for 16 and 24 h. Immunoblottings were performed as (A). Numbers indicate the expression ratios of LC3B-II/GAPDH. (C) PANC-1 cells were treated with GEF (25 μM) for 48 h, and electron microscopy was performed. Scale bars, 2 μm. N, nucleus; mit, mitochondria; AP, autophagosome; AL, autolysosome.
Figure Legend Snippet: Autophagy induction after treatment with GEF. (A) PANC-1 cells and BxPC-3 cells were treated with GEF (10 and 25 μM) for 16 and 24 h. Cellular proteins were separated by 15% SDS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPDH mAb was performed as an internal control. Numbers indicating the expression ratios of LC3B-II/GAPDH were determined using densitometry. (B) PANC-1 cells and BxPC-3 cells were cultured with or without GEF (25 μM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml), and pepstatin A (10 μg/ml) for 16 and 24 h. Immunoblottings were performed as (A). Numbers indicate the expression ratios of LC3B-II/GAPDH. (C) PANC-1 cells were treated with GEF (25 μM) for 48 h, and electron microscopy was performed. Scale bars, 2 μm. N, nucleus; mit, mitochondria; AP, autophagosome; AL, autolysosome.

Techniques Used: SDS Page, Expressing, Cell Culture, Electron Microscopy

13) Product Images from "Autophagy is a new protective mechanism against the cytotoxicity of platinum nanoparticles in human trophoblasts"

Article Title: Autophagy is a new protective mechanism against the cytotoxicity of platinum nanoparticles in human trophoblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41927-2

Autophagy activation by nPt in HchEpC1b cells, an EVT cell line. ( a ) Western blots of HchEpC1b cells, which were cultured with 25 μg/ml of nPt for 24 h with or without E64d (E64) and pepstatin A (P) for 2 h at the end of culture, were as follows: MAP1LC3B (LC3), SQSTM1, and ACTB. The expression levels of MAP1LC3B-II ( b ) or SQSTM1 ( c ) in the HchEpC1b cells cultured with nPt were shown in the graphs. Expression was normalized with ACTB levels. ( d ) Representative panels showed the merged images of anti-MAP1LC3B staining (LC3, green) and nuclei staining (DAPI, blue) of HchEpC1b cells cultured with 25 μg/ml of nPt for 24 h in the presence or absence of E64d and pepstatin for 2 h. ( e ) The graph showed the average number of LC3 puncta in HchEpC1b cells treated with the presence (black bars) or absence (white bars) of E64 and P, as shown in ( d ). ( f ) The cell number of HchEpC1b cells with nPt at the indicated concentrations (μg/ml) for 24 h were shown. The numbers of cells in the treatment groups were normalized to that with control treatment, PBS, as one. Data were expressed as the mean ± S.D. * p
Figure Legend Snippet: Autophagy activation by nPt in HchEpC1b cells, an EVT cell line. ( a ) Western blots of HchEpC1b cells, which were cultured with 25 μg/ml of nPt for 24 h with or without E64d (E64) and pepstatin A (P) for 2 h at the end of culture, were as follows: MAP1LC3B (LC3), SQSTM1, and ACTB. The expression levels of MAP1LC3B-II ( b ) or SQSTM1 ( c ) in the HchEpC1b cells cultured with nPt were shown in the graphs. Expression was normalized with ACTB levels. ( d ) Representative panels showed the merged images of anti-MAP1LC3B staining (LC3, green) and nuclei staining (DAPI, blue) of HchEpC1b cells cultured with 25 μg/ml of nPt for 24 h in the presence or absence of E64d and pepstatin for 2 h. ( e ) The graph showed the average number of LC3 puncta in HchEpC1b cells treated with the presence (black bars) or absence (white bars) of E64 and P, as shown in ( d ). ( f ) The cell number of HchEpC1b cells with nPt at the indicated concentrations (μg/ml) for 24 h were shown. The numbers of cells in the treatment groups were normalized to that with control treatment, PBS, as one. Data were expressed as the mean ± S.D. * p

Techniques Used: Activation Assay, Western Blot, Cell Culture, Expressing, Staining

14) Product Images from "Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1"

Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

Journal: Autophagy

doi: 10.1080/15548627.2015.1124223

TBC1D25 is not a substrate of starvation-induced autophagy. (A) Domain organization of TBC1D25 and sequestosome-1 (SQSTM1) and sequence alignment of the PB1-like domain of TBC1D25 and PB1 domain of SQSTM1. The N-terminal domains of TBC1D25 and SQSTM1 were aligned by using the HHpred software program (see also Fig. S2). Amino acid residues in the sequences that are identical and similar are shown against a black background and a shaded background, respectively. (B) MEF cells stably expressing EGFP-TBC1D25 were cultured for 1 h under nutrient-rich conditions (far left column) or starved conditions (right 3 columns). Note that EGFP-TBC1D25 and endogenous SQSTM1 colocalized well with LC3 dots under starved conditions. Scale bars: 10 μm. (C) The TBC1D25 protein level in both control MEF cells and  sqstm1 -KO MEF cells was unaltered by starvation. Control MEF cells and  sqstm1 -KO MEF cells were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 100 μM E64d and 100 μg/ml pepstatin A, and their cell lysates were analyzed by immunoblotting with anti-TBC1D25 antibody (top panel), anti-SQSTM1 antibody (middle panel), and anti-ACTB antibody (bottom panel). Note that SQSTM1, but not TBC1D25, was degraded by starvation. The asterisk indicates the nonspecific band of the anti-SQSTM1 antibody. The positions of the molecular mass markers (in kilodaltons) are shown on the left.
Figure Legend Snippet: TBC1D25 is not a substrate of starvation-induced autophagy. (A) Domain organization of TBC1D25 and sequestosome-1 (SQSTM1) and sequence alignment of the PB1-like domain of TBC1D25 and PB1 domain of SQSTM1. The N-terminal domains of TBC1D25 and SQSTM1 were aligned by using the HHpred software program (see also Fig. S2). Amino acid residues in the sequences that are identical and similar are shown against a black background and a shaded background, respectively. (B) MEF cells stably expressing EGFP-TBC1D25 were cultured for 1 h under nutrient-rich conditions (far left column) or starved conditions (right 3 columns). Note that EGFP-TBC1D25 and endogenous SQSTM1 colocalized well with LC3 dots under starved conditions. Scale bars: 10 μm. (C) The TBC1D25 protein level in both control MEF cells and sqstm1 -KO MEF cells was unaltered by starvation. Control MEF cells and sqstm1 -KO MEF cells were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 100 μM E64d and 100 μg/ml pepstatin A, and their cell lysates were analyzed by immunoblotting with anti-TBC1D25 antibody (top panel), anti-SQSTM1 antibody (middle panel), and anti-ACTB antibody (bottom panel). Note that SQSTM1, but not TBC1D25, was degraded by starvation. The asterisk indicates the nonspecific band of the anti-SQSTM1 antibody. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

Techniques Used: Sequencing, Software, Stable Transfection, Expressing, Cell Culture

15) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

16) Product Images from "Degradation of p47 by autophagy contributes to CADM1 overexpression in ATLL cells through the activation of NF-κB"

Article Title: Degradation of p47 by autophagy contributes to CADM1 overexpression in ATLL cells through the activation of NF-κB

Journal: Scientific Reports

doi: 10.1038/s41598-019-39424-7

p47 protein is efficiently degraded by the lysosome-dependent pathway in ATLL-related cell lines. ( A ) Immunoblot analysis of p47, CADM1, and NIK was performed in two HTLV-1-infected T-cell lines (MT2 and MT4) and two ATLL cell lines (KK1 and KOB) after treatment with or without MG132 for 48 hours. β-actin was used as a loading control. The cropped gels/blots are used in the figure, and the full-length gels/blots are presented in Supplementary Fig. S7 . ( B ) Immunoblot analysis of p47, CADM1, NEMO, and IκBα was performed in two HTLV-1-infected T-cell lines (MT2 and MT4) and two ATLL cell lines (KK1 and KOB) after treatment with or without E64d and pepstatin A for 48 hours. β-actin was used as a loading control. The cropped gels/blots are used in the figure, and the full-length gels/blots are presented in Supplementary Fig. S7 . ( C ) Relative expression of Beclin 1 was determined by quantitative RT-PCR in CD4 + T-cells from four healthy cases and leukemia cells from eight acute-type ATLL patients. The data represent the means ± S.D. of triplicate experiments and are presented relative to control CD4 + T-cells (lane 1). ** P
Figure Legend Snippet: p47 protein is efficiently degraded by the lysosome-dependent pathway in ATLL-related cell lines. ( A ) Immunoblot analysis of p47, CADM1, and NIK was performed in two HTLV-1-infected T-cell lines (MT2 and MT4) and two ATLL cell lines (KK1 and KOB) after treatment with or without MG132 for 48 hours. β-actin was used as a loading control. The cropped gels/blots are used in the figure, and the full-length gels/blots are presented in Supplementary Fig. S7 . ( B ) Immunoblot analysis of p47, CADM1, NEMO, and IκBα was performed in two HTLV-1-infected T-cell lines (MT2 and MT4) and two ATLL cell lines (KK1 and KOB) after treatment with or without E64d and pepstatin A for 48 hours. β-actin was used as a loading control. The cropped gels/blots are used in the figure, and the full-length gels/blots are presented in Supplementary Fig. S7 . ( C ) Relative expression of Beclin 1 was determined by quantitative RT-PCR in CD4 + T-cells from four healthy cases and leukemia cells from eight acute-type ATLL patients. The data represent the means ± S.D. of triplicate experiments and are presented relative to control CD4 + T-cells (lane 1). ** P

Techniques Used: Infection, Expressing, Quantitative RT-PCR

17) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

18) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

19) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

20) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

21) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

22) Product Images from "Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines"

Article Title: Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0164529

Macrolides induce cell death in amino acid-depleted culture by blocking autophagy flux. (A) Immunoblotting with LC3B and p62 Abs from the cell lysates of CAL 27 cells cultured in the complete culture medium with/without AZM, CAM (50 μM) in the presence or absence of lysosomal inhibitors (LIs) E-64d (10 μg/mL) and pepstatin A (10 μg/mL) for 24 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from PANC-1 cells treated with AZM for 24 hrs was used as a positive control [ 13 ]. (B) (upper panel) Immunoblotting of LC3 from the cell lysates of CAL 27 cells cultured in the complete culture medium or AAD culture medium containing 10% FBS with or without AZM (50 μM) and CAM (50 μM) for up to 60 min. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from PANC-1 cells treated with AZM (50 μM) for 24 hrs was used as a positive control [ 13 ]. (lower panel) The ratios of LC3B-II/GAPDH were plotted. (C) Cell growth inhibition of CAL 27, Detroit 562, and MEF cells treated with AZM (50 μM) and CAM (50 μM) in the complete culture medium (upper panel) and AAD culture medium (lower panel) for 24, 48, and 72 hrs. Viable cell numbers are expressed as percentage to viable cells cultured in the complete culture medium at each indicated culture period. Data are presented as means ± SEM. *p
Figure Legend Snippet: Macrolides induce cell death in amino acid-depleted culture by blocking autophagy flux. (A) Immunoblotting with LC3B and p62 Abs from the cell lysates of CAL 27 cells cultured in the complete culture medium with/without AZM, CAM (50 μM) in the presence or absence of lysosomal inhibitors (LIs) E-64d (10 μg/mL) and pepstatin A (10 μg/mL) for 24 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from PANC-1 cells treated with AZM for 24 hrs was used as a positive control [ 13 ]. (B) (upper panel) Immunoblotting of LC3 from the cell lysates of CAL 27 cells cultured in the complete culture medium or AAD culture medium containing 10% FBS with or without AZM (50 μM) and CAM (50 μM) for up to 60 min. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from PANC-1 cells treated with AZM (50 μM) for 24 hrs was used as a positive control [ 13 ]. (lower panel) The ratios of LC3B-II/GAPDH were plotted. (C) Cell growth inhibition of CAL 27, Detroit 562, and MEF cells treated with AZM (50 μM) and CAM (50 μM) in the complete culture medium (upper panel) and AAD culture medium (lower panel) for 24, 48, and 72 hrs. Viable cell numbers are expressed as percentage to viable cells cultured in the complete culture medium at each indicated culture period. Data are presented as means ± SEM. *p

Techniques Used: Blocking Assay, Cell Culture, Chick Chorioallantoic Membrane Assay, Derivative Assay, Positive Control, Inhibition

23) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

24) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

25) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

26) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

27) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

28) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

29) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

30) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

31) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

32) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

33) Product Images from "M-opsin protein degradation is inhibited by MG-132 in Rpe65−/− retinal explant culture"

Article Title: M-opsin protein degradation is inhibited by MG-132 in Rpe65−/− retinal explant culture

Journal: Molecular Vision

doi:

Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in  Rpe65 −/−  mice in retinal explants.  A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot.  B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads).  C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p
Figure Legend Snippet: Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in Rpe65 −/− mice in retinal explants. A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot. B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads). C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p

Techniques Used: Western Blot, Immunohistochemistry, Expressing, Protease Inhibitor, Mouse Assay, Staining, Labeling, Binding Assay

Expression of M-opsin protein in the retina after treatment with several types of protease inhibitors for 24 h in retinal explants. M-opsin protein levels were analyzed with western blotting after treatment with ( A ) pepstatin A, ( B ) E64d, and ( C ) MG-132.  D : Effect of MG-132 on M-opsin, S-opsin, GNAT2, GNAT1, and rhodopsin at the protein level analyzed with western blotting.  E : Quantitative analysis of M-opsin protein level.  F : Immunohistochemistry in MG-132-treated retinas for 24 h showed M-opsin (red). Cell nuclei were contrasted with DAPI (blue). M-opsin-positive cells increased after treatment with 2 μM or 10 μM MG-132 compared with vehicle in cone outer segments (arrows), ONL, and OPL (arrowheads) in the retina.  G : Densitometric analysis of immunofluorescence probed anti-M-opsin antibody in a 400-μm-wide section of retina. Histograms indicate mean±SD (n=6). *p
Figure Legend Snippet: Expression of M-opsin protein in the retina after treatment with several types of protease inhibitors for 24 h in retinal explants. M-opsin protein levels were analyzed with western blotting after treatment with ( A ) pepstatin A, ( B ) E64d, and ( C ) MG-132. D : Effect of MG-132 on M-opsin, S-opsin, GNAT2, GNAT1, and rhodopsin at the protein level analyzed with western blotting. E : Quantitative analysis of M-opsin protein level. F : Immunohistochemistry in MG-132-treated retinas for 24 h showed M-opsin (red). Cell nuclei were contrasted with DAPI (blue). M-opsin-positive cells increased after treatment with 2 μM or 10 μM MG-132 compared with vehicle in cone outer segments (arrows), ONL, and OPL (arrowheads) in the retina. G : Densitometric analysis of immunofluorescence probed anti-M-opsin antibody in a 400-μm-wide section of retina. Histograms indicate mean±SD (n=6). *p

Techniques Used: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

34) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

35) Product Images from "Autophagy regulates lipid metabolism through selective turnover of NCoR1"

Article Title: Autophagy regulates lipid metabolism through selective turnover of NCoR1

Journal: Nature Communications

doi: 10.1038/s41467-019-08829-3

Degradation of NCoR1 in autophagy-lysosomal pathway. a Immunoblot analysis. Both nuclear and cytoplasmic fractions were prepared from ATG7 -knockdown HepG2 cells under nutrient-rich and deprived conditions and subjected to immunoblotting with the indicated antibodies. Data are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analyses of cytoplasmic and nuclear NCoR1 relative to Gapdh and Lamin B, respectively. b Immunoblot analysis. HepG2 cells were cultured in the presence or absence of E64d and Pepstatin A (E.P.) for 24 h. Subsequently, both nuclear and cytoplasmic fractions were prepared from the HepG2 cells and subjected to immunoblotting with the indicated antibodies. Data are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analyses of cytoplasmic and nuclear NCoR1 relative to Gapdh and Lamin B, respectively. c Immunoblot analysis. GFP, wild-type ATG7, or ATG7 C572S was expressed in ATG7 -knockout HepG2 (#14) cells by adenovirus system. Forty-eight hours after infection, the cells were cultured under nutrient-rich or -deprived conditions. Thereafter, both nuclear and cytoplasmic fractions were prepared and subjected to immunoblotting with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analyses of cytoplasmic and nuclear NCoR1 relative to Gapdh and Lamin B, respectively. d Oxygen consumption rate (OCR). OCR of ATG7 −/− HepG2 cells (#14) expressing GFP or wild-type ATG7 in β-oxidation assay medium was measured using a Seahorse XF24 Extracellular Flux Analyzer. Etomoxir (final concentration 40 μM) was added to the cells after baseline measurement in assay medium. Arrows indicate the time when etomoxir was added to the cells. The graphs represent the average OCR at four time points. Data are means ± s.e.m. * P
Figure Legend Snippet: Degradation of NCoR1 in autophagy-lysosomal pathway. a Immunoblot analysis. Both nuclear and cytoplasmic fractions were prepared from ATG7 -knockdown HepG2 cells under nutrient-rich and deprived conditions and subjected to immunoblotting with the indicated antibodies. Data are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analyses of cytoplasmic and nuclear NCoR1 relative to Gapdh and Lamin B, respectively. b Immunoblot analysis. HepG2 cells were cultured in the presence or absence of E64d and Pepstatin A (E.P.) for 24 h. Subsequently, both nuclear and cytoplasmic fractions were prepared from the HepG2 cells and subjected to immunoblotting with the indicated antibodies. Data are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analyses of cytoplasmic and nuclear NCoR1 relative to Gapdh and Lamin B, respectively. c Immunoblot analysis. GFP, wild-type ATG7, or ATG7 C572S was expressed in ATG7 -knockout HepG2 (#14) cells by adenovirus system. Forty-eight hours after infection, the cells were cultured under nutrient-rich or -deprived conditions. Thereafter, both nuclear and cytoplasmic fractions were prepared and subjected to immunoblotting with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analyses of cytoplasmic and nuclear NCoR1 relative to Gapdh and Lamin B, respectively. d Oxygen consumption rate (OCR). OCR of ATG7 −/− HepG2 cells (#14) expressing GFP or wild-type ATG7 in β-oxidation assay medium was measured using a Seahorse XF24 Extracellular Flux Analyzer. Etomoxir (final concentration 40 μM) was added to the cells after baseline measurement in assay medium. Arrows indicate the time when etomoxir was added to the cells. The graphs represent the average OCR at four time points. Data are means ± s.e.m. * P

Techniques Used: Cell Culture, Knock-Out, Infection, Expressing, Oxidation Assay, Concentration Assay

36) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

37) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

38) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

39) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.
Figure Legend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

Techniques Used: Activity Assay, SDS Page, Marker

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P
Figure Legend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

Techniques Used: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

40) Product Images from "Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice"

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

Journal: Immunology

doi: 10.1046/j.1365-2567.2000.00000.x

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
Figure Legend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

Techniques Used: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.
Figure Legend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Techniques Used: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were
Figure Legend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunospot

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.
Figure Legend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Techniques Used: Mouse Assay

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.
Figure Legend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

Related Articles

Incubation:

Article Title: Partial Characterization of an Enzyme Fraction with Protease Activity Which Converts the Spore Peptidoglycan Hydrolase (SleC) Precursor to an Active Enzyme during Germination of Clostridium perfringens S40 Spores and Analysis of a Gene Cluster Involved in the Activity
Article Snippet: .. GSP was inhibited 68% after incubation at 30°C for 1 h with 1 mM PMSF and 20% with 0.1 mM PMSF but not with E-64 (0.1 mM), pepstatin A (0.1 mM), bestatin (0.1 mM), and EDTA (5 mM), suggesting that GSP belongs to a family of serine proteases. ..

Article Title: Suppression of Starvation-Induced Autophagy by Recombinant Toxic Shock Syndrome Toxin-1 in Epithelial Cells
Article Snippet: Autophagic induction, lysosomal staining and immunostaining Autophagy in HeLa 229 cells was induced by nutrient-starvation or rapamycin treatment. .. For nutrient-starvation, HeLa 229 cells were washed and incubated with Krebs Ringer bicarbonate buffer pH 7.6 (KRB; 118.5 mM NaCl, 4.47 mM KCl, 1.18 mM KH2 PO4 , 23.4 mM NaHCO3 , 6 mM glucose, 2.5 mM CaCl2 , 1.18 mM MgSO4 , and 6 mg/l phenol red) for 0–6 h. For rapamycin treatment, cells were incubated with 1 µM rapamycin [stock 1 mM in dimethyl sulfoxide (DMSO), Sigma Aldrich, St. Louis, MO] in MEM for 4 h. Lysosomal protease inhibitors, 10 µg/ml E64d (Peptide Institute, Inc., Osaka, Japan) and 10 µg/ml pepstatin A (Peptide Institute, Inc.) were used to inhibit the lysosomal turnover. .. For immunostaining, the cells were fixed with 4% paraformaldehyde (Wako), washed with PBS, and lysed with 50 µg/ml digitonin (Wako).

other:

Article Title: A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus
Article Snippet: Sixty hours after co‐infection, cells were harvested and lysed in buffer A containing 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 1 mM PMSF.

Article Title: The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers
Article Snippet: Leupeptin and pepstatin A were from Peptide Institute, Inc. (Osaka, Japan). l -Norleucine was purchased from MP Biomedicals, Inc. (Solon, OH).

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease
Article Snippet: Interestingly, in our study, Sap7 was not inhibited by pepstatin A ( ).

Concentration Assay:

Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice
Article Snippet: Pepstatin A (Peptide Institute, Osaka, Japan), a specific inhibitor of aspartyl proteases such as cathepsin D and pepsin, has previously been shown to cause prolonged inhibition of cathepsin D in mice, particularly in the spleen, liver and kidney. .. Pepstatin A was dissolved in dimethylsulphoxide (DMSO) and was further diluted in phosphate-buffered saline (PBS), at least 25 times, to avoid the toxic effect of a high concentration of DMSO. .. A DMSO control was included in the pepstatin A experiments.

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    Peptide Institute pepstatin a
    Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in  Rpe65 −/−  mice in retinal explants.  A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot.  B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads).  C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p
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    Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in  Rpe65 −/−  mice in retinal explants.  A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot.  B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads).  C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p

    Journal: Molecular Vision

    Article Title: M-opsin protein degradation is inhibited by MG-132 in Rpe65−/− retinal explant culture

    doi:

    Figure Lengend Snippet: Western blotting and immunohistochemistry analysis of M-opsin expression treated with a protease inhibitor mixture (20 μM pepstatin A, 30 μM E64d, and 10 μM MG-132) for 24 h in Rpe65 −/− mice in retinal explants. A : Immunoblots were performed using retina-RPE choroids treated with the protease inhibitor mixture. A loading control with GAPDH was included for each immunoblot. B : Immunolocalization of M-opsin protein in the retina treated with a protease inhibitor mixture. M-opsin was stained with polyclonal anti-M-opsin antibody (red), and cell nuclei were labeled with DNA-binding dye DAPI (blue). The number of immunoreactive products increased in the cone outer segments (arrows), ONL, and OPL (arrowheads). C : Histograms showing the area of immunoreactivity of M-opsin in a 400-μm-wide section of the retina. Data are expressed as the mean±SD (n=6). *p

    Article Snippet: Pepstatin A is an inhibitor of cathepsin D; E64d is an inhibitor of calpains and cathepsins B/H/L [ - ].

    Techniques: Western Blot, Immunohistochemistry, Expressing, Protease Inhibitor, Mouse Assay, Staining, Labeling, Binding Assay

    Expression of M-opsin protein in the retina after treatment with several types of protease inhibitors for 24 h in retinal explants. M-opsin protein levels were analyzed with western blotting after treatment with ( A ) pepstatin A, ( B ) E64d, and ( C ) MG-132.  D : Effect of MG-132 on M-opsin, S-opsin, GNAT2, GNAT1, and rhodopsin at the protein level analyzed with western blotting.  E : Quantitative analysis of M-opsin protein level.  F : Immunohistochemistry in MG-132-treated retinas for 24 h showed M-opsin (red). Cell nuclei were contrasted with DAPI (blue). M-opsin-positive cells increased after treatment with 2 μM or 10 μM MG-132 compared with vehicle in cone outer segments (arrows), ONL, and OPL (arrowheads) in the retina.  G : Densitometric analysis of immunofluorescence probed anti-M-opsin antibody in a 400-μm-wide section of retina. Histograms indicate mean±SD (n=6). *p

    Journal: Molecular Vision

    Article Title: M-opsin protein degradation is inhibited by MG-132 in Rpe65−/− retinal explant culture

    doi:

    Figure Lengend Snippet: Expression of M-opsin protein in the retina after treatment with several types of protease inhibitors for 24 h in retinal explants. M-opsin protein levels were analyzed with western blotting after treatment with ( A ) pepstatin A, ( B ) E64d, and ( C ) MG-132. D : Effect of MG-132 on M-opsin, S-opsin, GNAT2, GNAT1, and rhodopsin at the protein level analyzed with western blotting. E : Quantitative analysis of M-opsin protein level. F : Immunohistochemistry in MG-132-treated retinas for 24 h showed M-opsin (red). Cell nuclei were contrasted with DAPI (blue). M-opsin-positive cells increased after treatment with 2 μM or 10 μM MG-132 compared with vehicle in cone outer segments (arrows), ONL, and OPL (arrowheads) in the retina. G : Densitometric analysis of immunofluorescence probed anti-M-opsin antibody in a 400-μm-wide section of retina. Histograms indicate mean±SD (n=6). *p

    Article Snippet: Pepstatin A is an inhibitor of cathepsin D; E64d is an inhibitor of calpains and cathepsins B/H/L [ - ].

    Techniques: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

    Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

    Article Snippet: In contrast, treatment with pepstatin A, an aspartate protease inhibitor, resulted in marked alteration of Ii processing, indicating the accumulation of both LIP and SLIP Ii fragments in this experimental group.

    Techniques: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

    In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

    Article Snippet: In contrast, treatment with pepstatin A, an aspartate protease inhibitor, resulted in marked alteration of Ii processing, indicating the accumulation of both LIP and SLIP Ii fragments in this experimental group.

    Techniques: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

    T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

    Article Snippet: In contrast, treatment with pepstatin A, an aspartate protease inhibitor, resulted in marked alteration of Ii processing, indicating the accumulation of both LIP and SLIP Ii fragments in this experimental group.

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunospot

    The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

    Article Snippet: In contrast, treatment with pepstatin A, an aspartate protease inhibitor, resulted in marked alteration of Ii processing, indicating the accumulation of both LIP and SLIP Ii fragments in this experimental group.

    Techniques: Mouse Assay

    Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

    Article Snippet: In contrast, treatment with pepstatin A, an aspartate protease inhibitor, resulted in marked alteration of Ii processing, indicating the accumulation of both LIP and SLIP Ii fragments in this experimental group.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of nepenthesins by pepstatin and DAN ( A ) Pepstatin inhibition of nepenthesin I (•) at pH 3.0. Inhibition of porcine pepsin A (□) by pepstatin was also examined under the same conditions. ( B ) DAN inhibition of nepenthesin I in the presence (–•–) and absence (--•--) of cupric ions and of nepenthesin II in the presence (–○–) and absence (--○--) of cupric ions. Porcine pepsin A was treated with DAN in the same manner in the presence (–□–) and absence (--□--) of cupric ions.

    Journal: Biochemical Journal

    Article Title: Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases

    doi: 10.1042/BJ20031575

    Figure Lengend Snippet: Inhibition of nepenthesins by pepstatin and DAN ( A ) Pepstatin inhibition of nepenthesin I (•) at pH 3.0. Inhibition of porcine pepsin A (□) by pepstatin was also examined under the same conditions. ( B ) DAN inhibition of nepenthesin I in the presence (–•–) and absence (--•--) of cupric ions and of nepenthesin II in the presence (–○–) and absence (--○--) of cupric ions. Porcine pepsin A was treated with DAN in the same manner in the presence (–□–) and absence (--□--) of cupric ions.

    Article Snippet: Pepstatin A was from Peptide Institute (Osaka, Japan).

    Techniques: Inhibition

    N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

    Article Snippet: This modification of carbohydrate chains on the protein surface could act as a barrier to pepstatin A.

    Techniques: Activity Assay, SDS Page, Marker

    M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

    Article Snippet: This modification of carbohydrate chains on the protein surface could act as a barrier to pepstatin A.

    Techniques: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

    Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously  N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of  P. pastoris  transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. **  P

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

    Article Snippet: This modification of carbohydrate chains on the protein surface could act as a barrier to pepstatin A.

    Techniques: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay