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clasto-lactacystin β-lactone  (Millipore)

 
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    Millipore clasto-lactacystin β-lactone
    Clasto Lactacystin β Lactone, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clasto-lactacystin β-lactone/product/Millipore
    Average 90 stars, based on 1 article reviews
    clasto-lactacystin β-lactone - by Bioz Stars, 2026-02
    90/100 stars

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    API5 inhibits autophagic degradation of RIG‐I and MDA5 by phosphorylation. A and B) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected WT and API5 −/− A549 cells (A), and lungs from shCON and sh Api5 mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) or VSV (10 8.0 PFU mL −1 ; 60 µL per mouse) for 4 days (B). C and D) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected API5 −/− A549 cells reintroduced with empty vector (Vec.), API5 WT , or API5 S464A (C), and the lung tissues from Flag‐tagged API5 WT ‐, API5 S464A ‐ or empty vector (Vec.)‐overexpressing mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) for 4 days (D). E) Immunoblot analysis of extracts of API5 −/− A549 cells treated with bafilomycin A1 (BafA1; 200 n m ), chloroquine (CQ; 50 µM), 3‐methyladenine (3‐MA; 10 m m ), MG132 (10 µM) or <t>Lactacystin</t> (Lacta; 10 µ m ) for 6 h followed by infected with SeV for 12 h. (F) Immunoblot analysis of extracts of WT, API5 −/− , ATG5 −/− , API5 −/− ATG5 −/‐ HEK293T cells infected with SeV for 12 h. G) Immunoblot analysis of extracts of WT and ATG5 −/− HEK293T cells transfected with empty vector (Vec.) or plasmid expressing Flag‐API 5WT , or Flag‐API5 S464A followed by SeV infection for 12 h. Data are one representative of three biological replicates.
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    API5 inhibits autophagic degradation of RIG‐I and MDA5 by phosphorylation. A and B) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected WT and API5 −/− A549 cells (A), and lungs from shCON and sh Api5 mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) or VSV (10 8.0 PFU mL −1 ; 60 µL per mouse) for 4 days (B). C and D) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected API5 −/− A549 cells reintroduced with empty vector (Vec.), API5 WT , or API5 S464A (C), and the lung tissues from Flag‐tagged API5 WT ‐, API5 S464A ‐ or empty vector (Vec.)‐overexpressing mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) for 4 days (D). E) Immunoblot analysis of extracts of API5 −/− A549 cells treated with bafilomycin A1 (BafA1; 200 n m ), chloroquine (CQ; 50 µM), 3‐methyladenine (3‐MA; 10 m m ), MG132 (10 µM) or <t>Lactacystin</t> (Lacta; 10 µ m ) for 6 h followed by infected with SeV for 12 h. (F) Immunoblot analysis of extracts of WT, API5 −/− , ATG5 −/− , API5 −/− ATG5 −/‐ HEK293T cells infected with SeV for 12 h. G) Immunoblot analysis of extracts of WT and ATG5 −/− HEK293T cells transfected with empty vector (Vec.) or plasmid expressing Flag‐API 5WT , or Flag‐API5 S464A followed by SeV infection for 12 h. Data are one representative of three biological replicates.
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    API5 inhibits autophagic degradation of RIG‐I and MDA5 by phosphorylation. A and B) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected WT and API5 −/− A549 cells (A), and lungs from shCON and sh Api5 mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) or VSV (10 8.0 PFU mL −1 ; 60 µL per mouse) for 4 days (B). C and D) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected API5 −/− A549 cells reintroduced with empty vector (Vec.), API5 WT , or API5 S464A (C), and the lung tissues from Flag‐tagged API5 WT ‐, API5 S464A ‐ or empty vector (Vec.)‐overexpressing mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) for 4 days (D). E) Immunoblot analysis of extracts of API5 −/− A549 cells treated with bafilomycin A1 (BafA1; 200 n m ), chloroquine (CQ; 50 µM), 3‐methyladenine (3‐MA; 10 m m ), MG132 (10 µM) or <t>Lactacystin</t> (Lacta; 10 µ m ) for 6 h followed by infected with SeV for 12 h. (F) Immunoblot analysis of extracts of WT, API5 −/− , ATG5 −/− , API5 −/− ATG5 −/‐ HEK293T cells infected with SeV for 12 h. G) Immunoblot analysis of extracts of WT and ATG5 −/− HEK293T cells transfected with empty vector (Vec.) or plasmid expressing Flag‐API 5WT , or Flag‐API5 S464A followed by SeV infection for 12 h. Data are one representative of three biological replicates.
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    API5 inhibits autophagic degradation of RIG‐I and MDA5 by phosphorylation. A and B) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected WT and API5 −/− A549 cells (A), and lungs from shCON and sh Api5 mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) or VSV (10 8.0 PFU mL −1 ; 60 µL per mouse) for 4 days (B). C and D) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected API5 −/− A549 cells reintroduced with empty vector (Vec.), API5 WT , or API5 S464A (C), and the lung tissues from Flag‐tagged API5 WT ‐, API5 S464A ‐ or empty vector (Vec.)‐overexpressing mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) for 4 days (D). E) Immunoblot analysis of extracts of API5 −/− A549 cells treated with bafilomycin A1 (BafA1; 200 n m ), chloroquine (CQ; 50 µM), 3‐methyladenine (3‐MA; 10 m m ), MG132 (10 µM) or Lactacystin (Lacta; 10 µ m ) for 6 h followed by infected with SeV for 12 h. (F) Immunoblot analysis of extracts of WT, API5 −/− , ATG5 −/− , API5 −/− ATG5 −/‐ HEK293T cells infected with SeV for 12 h. G) Immunoblot analysis of extracts of WT and ATG5 −/− HEK293T cells transfected with empty vector (Vec.) or plasmid expressing Flag‐API 5WT , or Flag‐API5 S464A followed by SeV infection for 12 h. Data are one representative of three biological replicates.

    Journal: Advanced Science

    Article Title: API5 Phosphorylation Promotes Antiviral Immunity by Inhibiting Degradation of Cytosolic RNA Sensor RLRs

    doi: 10.1002/advs.202505479

    Figure Lengend Snippet: API5 inhibits autophagic degradation of RIG‐I and MDA5 by phosphorylation. A and B) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected WT and API5 −/− A549 cells (A), and lungs from shCON and sh Api5 mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) or VSV (10 8.0 PFU mL −1 ; 60 µL per mouse) for 4 days (B). C and D) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected API5 −/− A549 cells reintroduced with empty vector (Vec.), API5 WT , or API5 S464A (C), and the lung tissues from Flag‐tagged API5 WT ‐, API5 S464A ‐ or empty vector (Vec.)‐overexpressing mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) for 4 days (D). E) Immunoblot analysis of extracts of API5 −/− A549 cells treated with bafilomycin A1 (BafA1; 200 n m ), chloroquine (CQ; 50 µM), 3‐methyladenine (3‐MA; 10 m m ), MG132 (10 µM) or Lactacystin (Lacta; 10 µ m ) for 6 h followed by infected with SeV for 12 h. (F) Immunoblot analysis of extracts of WT, API5 −/− , ATG5 −/− , API5 −/− ATG5 −/‐ HEK293T cells infected with SeV for 12 h. G) Immunoblot analysis of extracts of WT and ATG5 −/− HEK293T cells transfected with empty vector (Vec.) or plasmid expressing Flag‐API 5WT , or Flag‐API5 S464A followed by SeV infection for 12 h. Data are one representative of three biological replicates.

    Article Snippet: The following antibodies were used in this study: Rabbit anti‐Flag Tag (0912), Rabbit anti‐His Tag (0812), Mouse anti‐GST Tag (EM80701), Mouse anti‐β‐Actin (M1210), Mouse anti‐Histone H3 (M1306), Rabbit anti‐IKKα/β (ET1611‐23), Rabbit anti‐p65 (ET1603‐12), and Rabbit anti‐ATG5 (ET1611‐38) were purchased by HuaAn Biotechnology; Mouse anti‐Flag Tag (F1804) was from Sigma–Aldrich; Rabbit anti‐GAPDH (AB‐P‐R001) was from GoodHere Technology; Mouse anti‐influenza viral proteins were prepared and stored in the laboratory; Rabbit anti‐API5 (ab65836) and Rabbit anti‐p62 (ab109012) were from Abcam; Mouse anti‐API5 (sc‐393341) and Mouse anti‐RIG‐I (sc‐376845) were from Santa Cruz; Rabbit anti‐SRPK1(A5854) and Rabbit anti‐MDA5 (A13645) were from ABclonal; Rabbit anti‐ RIG‐I (67556‐1‐Ig) and Rabbit anti‐IRF3 (11312‐1‐AP) were from Proteintech; Rabbit anti‐phospho‐IRF3 (13 786) was from Signalway Antibody; Rabbit anti‐MDA5 (5321), Mouse anti‐Ubiquitin (3936), Rabbit anti‐TBK1(3504), Rabbit anti‐phospho‐TBK1 (5483), Rabbit anti‐phospho‐IKKα/β (2697), Rabbit anti‐phospho‐p65 (3033), Rabbit anti‐phospho‐IκBα (2859) and Rabbit anti‐LC3B (2775) were purchased by Cell Signaling Technology; HRP‐conjugated goat‐anti mouse IgG (074‐1806) and HRP‐conjugated goat‐anti rabbit IgG (074‐1506) were from Kirkegaard & Perry Laboratories, Inc. Bafilomycin A1(BafA1; HY‐100558), chloroquine (CQ; HY‐17589A), 3‐methyladenine (3‐MA; HY‐19312), MG132 (HY‐13259), Lactacystin (Lacta; HY‐16594), SRPIN340 (HY‐13949,) and cycloheximide (CHX; HY‐12320) were purchased from MedChemExpress.

    Techniques: Phospho-proteomics, Western Blot, Infection, Plasmid Preparation, Transfection, Expressing