Structured Review

Carl Roth GmbH leupeptin
The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM <t>leupeptin</t> or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.
Leupeptin, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/leupeptin/product/Carl Roth GmbH
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
leupeptin - by Bioz Stars, 2020-07
92/100 stars

Images

1) Product Images from "The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain"

Article Title: The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121069

The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM leupeptin or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.
Figure Legend Snippet: The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM leupeptin or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.

Techniques Used: Cross-linking Immunoprecipitation, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Isolation, Mouse Assay, SDS Page, Molecular Weight

Related Articles

Concentration Assay:

Article Title: The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain
Article Snippet: .. For inhibition of proteases, the following reagents were used at the concentration indicated in : (Z-LL)2 -ketone (Peptanova), leupeptin (Roth), inhibitor X (EMD Millipore), and NH4 Cl (Merck). .. Protein extraction and immunoblotting.

Inhibition:

Article Title: The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain
Article Snippet: .. For inhibition of proteases, the following reagents were used at the concentration indicated in : (Z-LL)2 -ketone (Peptanova), leupeptin (Roth), inhibitor X (EMD Millipore), and NH4 Cl (Merck). .. Protein extraction and immunoblotting.

Western Blot:

Article Title: JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells
Article Snippet: .. Cell lysis and western blot analysis For the isolation of cellular proteins, confluent HEK cell cultures were lysed in 300 μl RIPA-lysis buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 15% glycerol, 1 mM NaF, and 1 mM Na3 VO4 ) supplemented with 10 μg/ml of each aprotinin, pepstatin (Sigma-Aldrich) and leupeptin (MP Biochemicals) as well as 0.8 μM Pefabloc (Carl Roth). ..

Isolation:

Article Title: JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells
Article Snippet: .. Cell lysis and western blot analysis For the isolation of cellular proteins, confluent HEK cell cultures were lysed in 300 μl RIPA-lysis buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 15% glycerol, 1 mM NaF, and 1 mM Na3 VO4 ) supplemented with 10 μg/ml of each aprotinin, pepstatin (Sigma-Aldrich) and leupeptin (MP Biochemicals) as well as 0.8 μM Pefabloc (Carl Roth). ..

Lysis:

Article Title: JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells
Article Snippet: .. Cell lysis and western blot analysis For the isolation of cellular proteins, confluent HEK cell cultures were lysed in 300 μl RIPA-lysis buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 15% glycerol, 1 mM NaF, and 1 mM Na3 VO4 ) supplemented with 10 μg/ml of each aprotinin, pepstatin (Sigma-Aldrich) and leupeptin (MP Biochemicals) as well as 0.8 μM Pefabloc (Carl Roth). ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Carl Roth GmbH leupeptin
    The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM <t>leupeptin</t> or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.
    Leupeptin, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptin/product/Carl Roth GmbH
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    leupeptin - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM leupeptin or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain

    doi: 10.1084/jem.20121069

    Figure Lengend Snippet: The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM leupeptin or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.

    Article Snippet: For inhibition of proteases, the following reagents were used at the concentration indicated in : (Z-LL)2 -ketone (Peptanova), leupeptin (Roth), inhibitor X (EMD Millipore), and NH4 Cl (Merck).

    Techniques: Cross-linking Immunoprecipitation, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Isolation, Mouse Assay, SDS Page, Molecular Weight