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rabbit anti atg5  (Novus Biologicals)


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    Structured Review

    Novus Biologicals rabbit anti atg5
    Rabbit Anti Atg5, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+atg5/ATG5+Antibody+-+BSA+Free/pm41968673-263-6-8
    Average 94 stars, based on 184 article reviews
    rabbit anti atg5 - by Bioz Stars, 2026-10
    94/100 stars

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    Related Articles

    Western Blot:

    Article Title: Disruption of Oligodendroglial Autophagy Leads to Myelin Morphological Deficits, Neuronal Apoptosis, and Cognitive Decline in Aged Mice
    Article Snippet: After blocking in 5% BSA (Sigma‐Aldrich) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C. .. The following antibodies were used for Western blot (WB) analysis: anti‐PLP (1:1000, rabbit, Abcam Cat# ab28486), anti‐MBP (1:2000, rat, Serotec Cat# MCA409S), anti‐Atg5 (1:1000; rabbit, Novus, Cat# NB110‐53818), anti‐CNPase (1:1000; rabbit, Cell Signaling, Cat# 5664), and anti‐α‐Tubulin, clone DM1A (1:10,000; mouse, Sigma‐Aldrich, Cat# T9026). .. After three 5‐min washes in TPBS (100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 N NaCl, 0.1% Tween‐20), membranes were incubated with secondary horseradish peroxidase‐conjugated antibodies (Millipore, Cat# AP308P, AP132P, AP189P) for 1 h at room temperature.

    Article Title: Disruption of Oligodendroglial Autophagy Leads to Myelin Morphological Deficits, Neuronal Apoptosis, and Cognitive Decline in Aged Mice.
    Article Snippet: After blocking in 5% BSA (Sigma- Aldrich) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C. .. The following antibodies were used for Western blot (WB) analysis: anti- PLP (1:1000, rabbit, Abcam Cat# ab28486), anti- MBP (1:2000, rat, Serotec Cat# MCA409S), anti- Atg5 (1:1000; rabbit, Novus, Cat# NB110- 53818), antiCNPase (1:1000; rabbit, Cell Signaling, Cat# 5664), and anti- αTubulin, clone DM1A (1:10,000; mouse, Sigma- Aldrich, Cat# T9026). .. After three 5- min washes in TPBS (100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 N NaCl, 0.1% Tween- 20), membranes were incubated with secondary horseradish peroxidase- conjugated antibodies (Millipore, Cat# AP308P, AP132P, AP189P) for 1 h at room temperature.

    Blocking Assay:

    Article Title: Deciphering melanophagy: role of the PTK2-ITCH-MLANA-OPTN cascade on melanophagy in melanocytes.
    Article Snippet: The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, 1620177). .. After blocking with 4% skim milk (MBcell, MBS1667) in TBST (25 mM Tris-base, 140 mM NaCl [GenDEPOT, T9200 and G0610], and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the membrane was incubated with indicated primary antibodies: anti-LC3 (NB100-2220; 1:3000) and anti-ATG5 (NB110-53818; 1:3000) antibodies, purchased from NOVUS biologicals; anti-ITCH (611198; 1:1000), obtained from BD Biosciences; anti-MYC (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; antiOPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5-15257, 1:1000), purchased from Invitrogen; anti-ACTB/β-Actin (660009-1-Ig; 1:10000) were purchased from Proteintech. .. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).

    Article Title: Deciphering melanophagy: role of the PTK2-ITCH-MLANA-OPTN cascade on melanophagy in melanocytes
    Article Snippet: The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad 1,620,177). .. After blocking with 4% skim milk (MBcell, MB-S1667) in TBST (25 mm Tris-base, 140 mm NaCl [GenDEPOT, T9200 and G0610], and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the membrane was incubated with indicated primary antibodies: anti-LC3 (NB100–2220; 1:3000) and anti-ATG5 (NB110–53818; 1:3000) antibodies, purchased from NOVUS biologicals; anti-ITCH (611198; 1:1000), obtained from BD Biosciences; anti-MYC (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; anti-OPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5–15257, 1:1000), purchased from Invitrogen; anti-ACTB/β-Actin (660009–1-Ig; 1:10000) were purchased from Proteintech. .. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).

    Membrane:

    Article Title: Deciphering melanophagy: role of the PTK2-ITCH-MLANA-OPTN cascade on melanophagy in melanocytes.
    Article Snippet: The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, 1620177). .. After blocking with 4% skim milk (MBcell, MBS1667) in TBST (25 mM Tris-base, 140 mM NaCl [GenDEPOT, T9200 and G0610], and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the membrane was incubated with indicated primary antibodies: anti-LC3 (NB100-2220; 1:3000) and anti-ATG5 (NB110-53818; 1:3000) antibodies, purchased from NOVUS biologicals; anti-ITCH (611198; 1:1000), obtained from BD Biosciences; anti-MYC (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; antiOPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5-15257, 1:1000), purchased from Invitrogen; anti-ACTB/β-Actin (660009-1-Ig; 1:10000) were purchased from Proteintech. .. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).

    Article Title: Deciphering melanophagy: role of the PTK2-ITCH-MLANA-OPTN cascade on melanophagy in melanocytes
    Article Snippet: The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad 1,620,177). .. After blocking with 4% skim milk (MBcell, MB-S1667) in TBST (25 mm Tris-base, 140 mm NaCl [GenDEPOT, T9200 and G0610], and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the membrane was incubated with indicated primary antibodies: anti-LC3 (NB100–2220; 1:3000) and anti-ATG5 (NB110–53818; 1:3000) antibodies, purchased from NOVUS biologicals; anti-ITCH (611198; 1:1000), obtained from BD Biosciences; anti-MYC (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; anti-OPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5–15257, 1:1000), purchased from Invitrogen; anti-ACTB/β-Actin (660009–1-Ig; 1:10000) were purchased from Proteintech. .. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).

    Incubation:

    Article Title: Deciphering melanophagy: role of the PTK2-ITCH-MLANA-OPTN cascade on melanophagy in melanocytes.
    Article Snippet: The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, 1620177). .. After blocking with 4% skim milk (MBcell, MBS1667) in TBST (25 mM Tris-base, 140 mM NaCl [GenDEPOT, T9200 and G0610], and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the membrane was incubated with indicated primary antibodies: anti-LC3 (NB100-2220; 1:3000) and anti-ATG5 (NB110-53818; 1:3000) antibodies, purchased from NOVUS biologicals; anti-ITCH (611198; 1:1000), obtained from BD Biosciences; anti-MYC (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; antiOPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5-15257, 1:1000), purchased from Invitrogen; anti-ACTB/β-Actin (660009-1-Ig; 1:10000) were purchased from Proteintech. .. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).

    Article Title: The glucose metabolism reprogramming of yak Sertoli cells under hypoxia is regulated by autophagy.
    Article Snippet: .. Next, 1% BSA was mixed in the cell for 2 h. And cells were separately incubated with anti-Sox9 (1:250 dilution, Bioss, Beijing, China), anti-Atg5 (1:500 dilution, NOVUS, Littleton, US), anti-Beclin1 (1:500 dilution, Prepared by our laboratory, China), anti-P62 (1:500 dilution, Prepared by our laboratory, China), anti-LC3 (1:500 dilution, Prepared by our laboratory, China), anti-GLUT1 (1:250 dilution, Proteintech, Wuhan, China), anti-GLUT3 (1:250 dilution, Bioss, Beijing, China), anti-GLUT8 (1:250 dilution, Bioss, Beijing, China), anti-MCT1 (1:250 dilution, Bioss, Beijing, China), and anti-MCT4 (1:250 dilution, Bioss, Beijing, China) overnight at 4 °C. .. Subsequently, cells were paired with secondary antibody Alexa Fluor 594 labeled Goat anti-rabbit IgG (1:500 diluted; Bioss, Beijing, China) incubated for 1 h. Washed each step with PBS 3 times before restaining with DAPI (4’ −6-diamidine-2-phenylindole) for 3 min.

    Article Title: Deciphering melanophagy: role of the PTK2-ITCH-MLANA-OPTN cascade on melanophagy in melanocytes
    Article Snippet: The samples were then separated using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad 1,620,177). .. After blocking with 4% skim milk (MBcell, MB-S1667) in TBST (25 mm Tris-base, 140 mm NaCl [GenDEPOT, T9200 and G0610], and 0.05% Tween® 20 [Sigma-Aldrich, P7949]), the membrane was incubated with indicated primary antibodies: anti-LC3 (NB100–2220; 1:3000) and anti-ATG5 (NB110–53818; 1:3000) antibodies, purchased from NOVUS biologicals; anti-ITCH (611198; 1:1000), obtained from BD Biosciences; anti-MYC (SC-40; 1:1000), anti-GFP (SC-9996; 1:1000), anti-HA (SC-7392; 1:1000), anti-ubiquitin (SC-8017; 1:1000), anti-MLANA (SC-20032; 1:3000), purchased from Santa Cruz Biotechnology; anti-DYDDDDK (2368; 1:3000), anti-PTK2 (3285; 1:1000), anti-phospho-PTK2 Tyr397 (3283; 1:1000), obtained from Cell Signaling Technology; anti-OPTN (ab23666; 1:1000), purchased from Abcam; anti-RFP (MA5–15257, 1:1000), purchased from Invitrogen; anti-ACTB/β-Actin (660009–1-Ig; 1:10000) were purchased from Proteintech. .. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076 and 7074; 1:5000).

    Article Title: The glucose metabolism reprogramming of yak Sertoli cells under hypoxia is regulated by autophagy
    Article Snippet: .. Next, 1% BSA was mixed in the cell for 2 h. And cells were separately incubated with anti-Sox9 (1:250 dilution, Bioss, Beijing, China), anti-Atg5 (1:500 dilution, NOVUS, Littleton, US), anti-Beclin1 (1:500 dilution, Prepared by our laboratory, China), anti-P62 (1:500 dilution, Prepared by our laboratory, China), anti-LC3 (1:500 dilution, Prepared by our laboratory, China), anti-GLUT1 (1:250 dilution, Proteintech, Wuhan, China), anti-GLUT3 (1:250 dilution, Bioss, Beijing, China), anti-GLUT8 (1:250 dilution, Bioss, Beijing, China), anti-MCT1 (1:250 dilution, Bioss, Beijing, China), and anti-MCT4 (1:250 dilution, Bioss, Beijing, China) overnight at 4 °C. .. Subsequently, cells were paired with secondary antibody Alexa Fluor 594 labeled Goat anti-rabbit IgG (1:500 diluted; Bioss, Beijing, China) incubated for 1 h. Washed each step with PBS 3 times before restaining with DAPI (4' −6-diamidine-2-phenylindole) for 3 min.

    Plasmid Purification:

    Article Title: Disruption of Oligodendroglial Autophagy Leads to Myelin Morphological Deficits, Neuronal Apoptosis, and Cognitive Decline in Aged Mice.
    Article Snippet: After blocking in 5% BSA (Sigma- Aldrich) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C. .. The following antibodies were used for Western blot (WB) analysis: anti- PLP (1:1000, rabbit, Abcam Cat# ab28486), anti- MBP (1:2000, rat, Serotec Cat# MCA409S), anti- Atg5 (1:1000; rabbit, Novus, Cat# NB110- 53818), antiCNPase (1:1000; rabbit, Cell Signaling, Cat# 5664), and anti- αTubulin, clone DM1A (1:10,000; mouse, Sigma- Aldrich, Cat# T9026). .. After three 5- min washes in TPBS (100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 N NaCl, 0.1% Tween- 20), membranes were incubated with secondary horseradish peroxidase- conjugated antibodies (Millipore, Cat# AP308P, AP132P, AP189P) for 1 h at room temperature.



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    Molecular interaction between SDE2 and <t>ATG5.</t> (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
    Atg5, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    atg5  (Santa Cruz Biotechnology)
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    Molecular interaction between SDE2 and <t>ATG5.</t> (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
    Atg5, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular interaction between SDE2 and <t>ATG5.</t> (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
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    atg5 d5f5u rabbit mab  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc atg5 d5f5u rabbit mab
    Molecular interaction between SDE2 and <t>ATG5.</t> (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
    Atg5 D5f5u Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+atg5/Atg5+Rabbit+mAb/pm41857115-129-39-55
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    anti atg5  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc anti atg5
    Molecular interaction between SDE2 and <t>ATG5.</t> (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
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    De novo HSV-2 infection induces IFI16-cGAS and IFI16-STING-dependent IRF3 activation, and the crosstalk of type I interferon pathway with inflammasome and autophagy pathways, as well as dynamic ubiquitination and acetylation of cGAS. HFF cells were left uninfected or infected with HSV-2 at 1 MOI for 4, 8 or 24h. (A) Equal amounts of whole cell lysate proteins were IPed with anti-cGAS antibody and western blotted for NLRP3, IFI16, K-63 linkage-specific polyubiquitin, Beclin-1 and Caspase-1 (pro and cleaved) ( <xref ref-type=Table 1 ). Fold changes are included at the bottom of the respective blots. (B) Equal amounts of whole cell lysate proteins were IPed with anti-STING antibody and western blotted for NLRP3, IFI16, cGAS, ASC, pro-Caspase-1 and LC3 IIA/B. Equal amounts of whole cell lysate proteins were IPed with anti-Acetylated Lysine antibody and western blotted for IFI16 and cGAS. Red boxes are used to identify bands. (C) Equal amounts of whole cell lysate proteins were IPed with antibodies against the proteins found to interact with STING and western blotted for STING or IPed with antibodies against cGAS or IFI16 and western blotted for Acetylated Lysine. (D) Levels of total NLRP3, IFI16, cGAS, AIM2, ASC, Caspase-1 (pro and cleaved), IL-1β (pro and cleaved), IL-18 (pro- and cleaved), Gasdermin-D (pro and cleaved), STING, pSTING, IRF3, pIRF3, ATG5, Beclin-1, LC3 IIA/B and Tubulin (as loading control) were detected with their respective antibodies. Fold changes are included at the bottom of the respective blots. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

    doi: 10.3389/fimmu.2026.1717787

    Figure Lengend Snippet: De novo HSV-2 infection induces IFI16-cGAS and IFI16-STING-dependent IRF3 activation, and the crosstalk of type I interferon pathway with inflammasome and autophagy pathways, as well as dynamic ubiquitination and acetylation of cGAS. HFF cells were left uninfected or infected with HSV-2 at 1 MOI for 4, 8 or 24h. (A) Equal amounts of whole cell lysate proteins were IPed with anti-cGAS antibody and western blotted for NLRP3, IFI16, K-63 linkage-specific polyubiquitin, Beclin-1 and Caspase-1 (pro and cleaved) ( Table 1 ). Fold changes are included at the bottom of the respective blots. (B) Equal amounts of whole cell lysate proteins were IPed with anti-STING antibody and western blotted for NLRP3, IFI16, cGAS, ASC, pro-Caspase-1 and LC3 IIA/B. Equal amounts of whole cell lysate proteins were IPed with anti-Acetylated Lysine antibody and western blotted for IFI16 and cGAS. Red boxes are used to identify bands. (C) Equal amounts of whole cell lysate proteins were IPed with antibodies against the proteins found to interact with STING and western blotted for STING or IPed with antibodies against cGAS or IFI16 and western blotted for Acetylated Lysine. (D) Levels of total NLRP3, IFI16, cGAS, AIM2, ASC, Caspase-1 (pro and cleaved), IL-1β (pro and cleaved), IL-18 (pro- and cleaved), Gasdermin-D (pro and cleaved), STING, pSTING, IRF3, pIRF3, ATG5, Beclin-1, LC3 IIA/B and Tubulin (as loading control) were detected with their respective antibodies. Fold changes are included at the bottom of the respective blots.

    Article Snippet: ATG5 , Rabbit polyclonal , Affinity Biosciences, Cincinnati, Ohio , DF6010.

    Techniques: Infection, Activation Assay, Ubiquitin Proteomics, Western Blot, Control

    Molecular interaction between SDE2 and ATG5. (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Molecular interaction between SDE2 and ATG5. (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Expressing, Clinical Proteomics, Western Blot, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Ubiquitin Proteomics, Mutagenesis, Control

    Impact of SDE2 knockdown on cellular metabolism and autophagy in MM cells. (A) Fluorescence imaging of CFDA/DAPI-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (B) Fluorescence imaging of acridine orange-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (C) MitoTracker staining to evaluate mitochondrial activity in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (D) Transmission electron microscopy (TEM) images showing autophagosomes (indicated by red arrows) and autolysosomes (indicated by yellow arrows) in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (E) Western blot analysis of p62 and ATG5 expression levels in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (F) mRFP-GFP-LC3 dual fluorescence labeling of OPM-2 and KMS-11 cells with or without SDE2 knockdown, visualizing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). The bar graphs quantify the total number of puncta and the relative proportion of autophagosomes to autolysosomes. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Impact of SDE2 knockdown on cellular metabolism and autophagy in MM cells. (A) Fluorescence imaging of CFDA/DAPI-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (B) Fluorescence imaging of acridine orange-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (C) MitoTracker staining to evaluate mitochondrial activity in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (D) Transmission electron microscopy (TEM) images showing autophagosomes (indicated by red arrows) and autolysosomes (indicated by yellow arrows) in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (E) Western blot analysis of p62 and ATG5 expression levels in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (F) mRFP-GFP-LC3 dual fluorescence labeling of OPM-2 and KMS-11 cells with or without SDE2 knockdown, visualizing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). The bar graphs quantify the total number of puncta and the relative proportion of autophagosomes to autolysosomes. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Knockdown, Fluorescence, Imaging, Staining, Activity Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Labeling

    SDE2 promotes MM cell proliferation via its UBL domain–dependent inhibition of ferroptosis and autophagy. (A) Colony formation assays were performed in H929 cells transfected with empty vector, SDE2, or SDE2Δ1. Representative images (left) and quantification of colony numbers (right) are shown. (B) Intracellular glutathione (GSH), ferrous iron (Fe 2+ ), and malondialdehyde (MDA) levels were measured using corresponding colorimetric kits. (C) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in the indicated groups; GAPDH was used as a loading control. Densitometric quantification is shown on the right. (D) Lipid peroxidation was assessed by BODIPY 581/591 C11 staining in H929 cells expressing vector, SDE2, or SDE2Δ1. Representative fluorescence images of oxidized (green) and non-oxidized (red) signals are shown. (E) Quantification of oxidized BODIPY fluorescence intensity from (D). (F) Quantification of MitoTracker Red fluorescence intensity in the indicated groups. (G) Mitochondrial morphology was visualized by MitoTracker Red staining in H929 cells. (H) Autophagic flux was evaluated using the mRFP-GFP-LC3 tandem fluorescent-tagged reporter system. Representative images show red (mRFP), green (GFP), and merged puncta. (I) Quantification of LC3 puncta number and percentage of mRFP + /GFP − autolysosomes per cell from (H). (J) Western blot analysis of autophagy-related proteins p62, ATG5, and ATG7 in the indicated groups. GAPDH served as a loading control. Densitometric quantification is shown below. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: SDE2 promotes MM cell proliferation via its UBL domain–dependent inhibition of ferroptosis and autophagy. (A) Colony formation assays were performed in H929 cells transfected with empty vector, SDE2, or SDE2Δ1. Representative images (left) and quantification of colony numbers (right) are shown. (B) Intracellular glutathione (GSH), ferrous iron (Fe 2+ ), and malondialdehyde (MDA) levels were measured using corresponding colorimetric kits. (C) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in the indicated groups; GAPDH was used as a loading control. Densitometric quantification is shown on the right. (D) Lipid peroxidation was assessed by BODIPY 581/591 C11 staining in H929 cells expressing vector, SDE2, or SDE2Δ1. Representative fluorescence images of oxidized (green) and non-oxidized (red) signals are shown. (E) Quantification of oxidized BODIPY fluorescence intensity from (D). (F) Quantification of MitoTracker Red fluorescence intensity in the indicated groups. (G) Mitochondrial morphology was visualized by MitoTracker Red staining in H929 cells. (H) Autophagic flux was evaluated using the mRFP-GFP-LC3 tandem fluorescent-tagged reporter system. Representative images show red (mRFP), green (GFP), and merged puncta. (I) Quantification of LC3 puncta number and percentage of mRFP + /GFP − autolysosomes per cell from (H). (J) Western blot analysis of autophagy-related proteins p62, ATG5, and ATG7 in the indicated groups. GAPDH served as a loading control. Densitometric quantification is shown below. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Inhibition, Transfection, Plasmid Preparation, Western Blot, Control, Staining, Expressing, Fluorescence

    Modulation of autophagy and ferroptosis in MM cells through SDE2 manipulation and pharmacological interventions. KMS-11 cells treated with SDE2 knockdown and/or the autophagy inhibitor chloroquine (CQ): (A) Western blot analysis of autophagy-related proteins LC3 and p62 in KMS-11 cells. Densitometric quantification is shown on the right. (B) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells under the same treatments as in (A), with corresponding quantification on the right. (C) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. H929 cells treated with SDE2 overexpression and/or the ferroptosis inducer Erastin: (D) Western blot analysis of autophagy-related proteins LC3 and p62 in H929 cells. Densitometric quantification is shown on the right. (E) Western blot analysis and quantification of ATG5 and ATG7 in H929 cells under the same treatments as in (D). (F) mRFP-GFP-LC3 dual fluorescence labeling visualizing autophagic flux in H929 cells, showing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). KMS-11 cells treated with SDE2 knockdown and/or ATG5 knockdown: (G) Western blot analysis and quantification of Beclin1 and p62 in KMS-11 cells expressing shCtrl or shSDE2, with or without ATG5 knockdown. (H) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells with single or combined knockdown of SDE2 and ATG5. Quantification is shown on the right. (I) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Modulation of autophagy and ferroptosis in MM cells through SDE2 manipulation and pharmacological interventions. KMS-11 cells treated with SDE2 knockdown and/or the autophagy inhibitor chloroquine (CQ): (A) Western blot analysis of autophagy-related proteins LC3 and p62 in KMS-11 cells. Densitometric quantification is shown on the right. (B) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells under the same treatments as in (A), with corresponding quantification on the right. (C) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. H929 cells treated with SDE2 overexpression and/or the ferroptosis inducer Erastin: (D) Western blot analysis of autophagy-related proteins LC3 and p62 in H929 cells. Densitometric quantification is shown on the right. (E) Western blot analysis and quantification of ATG5 and ATG7 in H929 cells under the same treatments as in (D). (F) mRFP-GFP-LC3 dual fluorescence labeling visualizing autophagic flux in H929 cells, showing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). KMS-11 cells treated with SDE2 knockdown and/or ATG5 knockdown: (G) Western blot analysis and quantification of Beclin1 and p62 in KMS-11 cells expressing shCtrl or shSDE2, with or without ATG5 knockdown. (H) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells with single or combined knockdown of SDE2 and ATG5. Quantification is shown on the right. (I) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Knockdown, Western Blot, Over Expression, Fluorescence, Labeling, Expressing

    Functional interplay between ATG5 and SDE2 in MM progression and ferroptosis regulation. (A) Western blot analysis of ATG5 expression in malignant cells (T) and non-malignant bone marrow cells (N) isolated from the peripheral blood of MM patients. (B) Western blot analysis confirming the efficiency of ATG5 overexpression in OPM-2 and H929 cells transfected with a plasmid encoding the full-length ATG5 sequence. (C) Crystal violet staining of Transwell migration assays comparing the migratory ability of OPM-2 and H929 cells with or without ATG5 overexpression. (D) Experimental setup for H929 cells treated with SDE2 overexpression, ATG5 overexpression, or a combination of both. (E) Western blot analysis of SDE2 and ATG5 protein levels in H929 cells from the four groups defined in (D). GAPDH was used as a loading control. (F) (Top) Colony formation assay evaluating the proliferative capacity of H929 cells under different treatments. (Bottom) Transwell assay assessing the migratory ability of H929 cells under different treatments. (G) Biochemical analysis of ferroptosis-related markers (MDA, GSH, and Fe 2+ ) in H929 cells subjected to different treatments. (H) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in H929 cells under different treatments. (I) Western blot analysis showing the effects of different treatments on LC3 expression and p62 degradation in H929 cells. (J) Representative images of tumors formed 35 days after subcutaneous inoculation of differently treated H929 stable cell lines into nude mice. (K) Tumor growth curves showing volume changes of tumors derived from differently treated H929 cell lines over time. (L) Average tumor weights of tumors derived from differently treated H929 cell lines. (M) Western blot analysis of NCOA4, ACSL4, FTH1, and GPX4 expression levels in tumors derived from differently treated H929 cell lines. (N) Western blot analysis showing the activation levels of the ATG5/ATG7 pathway and p62 degradation in tumors derived from differently treated H929 cell lines. (O) Representative images of H&E staining and Ki67 immunohistochemical staining of tumor sections derived from differently treated H929 cell lines. The bar graph quantifies the percentage of proliferating cells (Ki67-positive). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Functional interplay between ATG5 and SDE2 in MM progression and ferroptosis regulation. (A) Western blot analysis of ATG5 expression in malignant cells (T) and non-malignant bone marrow cells (N) isolated from the peripheral blood of MM patients. (B) Western blot analysis confirming the efficiency of ATG5 overexpression in OPM-2 and H929 cells transfected with a plasmid encoding the full-length ATG5 sequence. (C) Crystal violet staining of Transwell migration assays comparing the migratory ability of OPM-2 and H929 cells with or without ATG5 overexpression. (D) Experimental setup for H929 cells treated with SDE2 overexpression, ATG5 overexpression, or a combination of both. (E) Western blot analysis of SDE2 and ATG5 protein levels in H929 cells from the four groups defined in (D). GAPDH was used as a loading control. (F) (Top) Colony formation assay evaluating the proliferative capacity of H929 cells under different treatments. (Bottom) Transwell assay assessing the migratory ability of H929 cells under different treatments. (G) Biochemical analysis of ferroptosis-related markers (MDA, GSH, and Fe 2+ ) in H929 cells subjected to different treatments. (H) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in H929 cells under different treatments. (I) Western blot analysis showing the effects of different treatments on LC3 expression and p62 degradation in H929 cells. (J) Representative images of tumors formed 35 days after subcutaneous inoculation of differently treated H929 stable cell lines into nude mice. (K) Tumor growth curves showing volume changes of tumors derived from differently treated H929 cell lines over time. (L) Average tumor weights of tumors derived from differently treated H929 cell lines. (M) Western blot analysis of NCOA4, ACSL4, FTH1, and GPX4 expression levels in tumors derived from differently treated H929 cell lines. (N) Western blot analysis showing the activation levels of the ATG5/ATG7 pathway and p62 degradation in tumors derived from differently treated H929 cell lines. (O) Representative images of H&E staining and Ki67 immunohistochemical staining of tumor sections derived from differently treated H929 cell lines. The bar graph quantifies the percentage of proliferating cells (Ki67-positive). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Functional Assay, Western Blot, Expressing, Isolation, Over Expression, Transfection, Plasmid Preparation, Sequencing, Staining, Migration, Control, Colony Assay, Transwell Assay, Stable Transfection, Derivative Assay, Activation Assay, Immunohistochemical staining

    Molecular interaction between SDE2 and ATG5. (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Molecular interaction between SDE2 and ATG5. (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Expressing, Clinical Proteomics, Western Blot, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Ubiquitin Proteomics, Mutagenesis, Control

    Impact of SDE2 knockdown on cellular metabolism and autophagy in MM cells. (A) Fluorescence imaging of CFDA/DAPI-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (B) Fluorescence imaging of acridine orange-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (C) MitoTracker staining to evaluate mitochondrial activity in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (D) Transmission electron microscopy (TEM) images showing autophagosomes (indicated by red arrows) and autolysosomes (indicated by yellow arrows) in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (E) Western blot analysis of p62 and ATG5 expression levels in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (F) mRFP-GFP-LC3 dual fluorescence labeling of OPM-2 and KMS-11 cells with or without SDE2 knockdown, visualizing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). The bar graphs quantify the total number of puncta and the relative proportion of autophagosomes to autolysosomes. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Impact of SDE2 knockdown on cellular metabolism and autophagy in MM cells. (A) Fluorescence imaging of CFDA/DAPI-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (B) Fluorescence imaging of acridine orange-stained OPM-2 and KMS-11 cells with or without SDE2 knockdown (left). The bar graph quantifies relative fluorescence intensity (right). (C) MitoTracker staining to evaluate mitochondrial activity in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (D) Transmission electron microscopy (TEM) images showing autophagosomes (indicated by red arrows) and autolysosomes (indicated by yellow arrows) in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (E) Western blot analysis of p62 and ATG5 expression levels in OPM-2 and KMS-11 cells with or without SDE2 knockdown. (F) mRFP-GFP-LC3 dual fluorescence labeling of OPM-2 and KMS-11 cells with or without SDE2 knockdown, visualizing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). The bar graphs quantify the total number of puncta and the relative proportion of autophagosomes to autolysosomes. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Knockdown, Fluorescence, Imaging, Staining, Activity Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Labeling

    SDE2 promotes MM cell proliferation via its UBL domain–dependent inhibition of ferroptosis and autophagy. (A) Colony formation assays were performed in H929 cells transfected with empty vector, SDE2, or SDE2Δ1. Representative images (left) and quantification of colony numbers (right) are shown. (B) Intracellular glutathione (GSH), ferrous iron (Fe 2+ ), and malondialdehyde (MDA) levels were measured using corresponding colorimetric kits. (C) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in the indicated groups; GAPDH was used as a loading control. Densitometric quantification is shown on the right. (D) Lipid peroxidation was assessed by BODIPY 581/591 C11 staining in H929 cells expressing vector, SDE2, or SDE2Δ1. Representative fluorescence images of oxidized (green) and non-oxidized (red) signals are shown. (E) Quantification of oxidized BODIPY fluorescence intensity from (D). (F) Quantification of MitoTracker Red fluorescence intensity in the indicated groups. (G) Mitochondrial morphology was visualized by MitoTracker Red staining in H929 cells. (H) Autophagic flux was evaluated using the mRFP-GFP-LC3 tandem fluorescent-tagged reporter system. Representative images show red (mRFP), green (GFP), and merged puncta. (I) Quantification of LC3 puncta number and percentage of mRFP + /GFP − autolysosomes per cell from (H). (J) Western blot analysis of autophagy-related proteins p62, ATG5, and ATG7 in the indicated groups. GAPDH served as a loading control. Densitometric quantification is shown below. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: SDE2 promotes MM cell proliferation via its UBL domain–dependent inhibition of ferroptosis and autophagy. (A) Colony formation assays were performed in H929 cells transfected with empty vector, SDE2, or SDE2Δ1. Representative images (left) and quantification of colony numbers (right) are shown. (B) Intracellular glutathione (GSH), ferrous iron (Fe 2+ ), and malondialdehyde (MDA) levels were measured using corresponding colorimetric kits. (C) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in the indicated groups; GAPDH was used as a loading control. Densitometric quantification is shown on the right. (D) Lipid peroxidation was assessed by BODIPY 581/591 C11 staining in H929 cells expressing vector, SDE2, or SDE2Δ1. Representative fluorescence images of oxidized (green) and non-oxidized (red) signals are shown. (E) Quantification of oxidized BODIPY fluorescence intensity from (D). (F) Quantification of MitoTracker Red fluorescence intensity in the indicated groups. (G) Mitochondrial morphology was visualized by MitoTracker Red staining in H929 cells. (H) Autophagic flux was evaluated using the mRFP-GFP-LC3 tandem fluorescent-tagged reporter system. Representative images show red (mRFP), green (GFP), and merged puncta. (I) Quantification of LC3 puncta number and percentage of mRFP + /GFP − autolysosomes per cell from (H). (J) Western blot analysis of autophagy-related proteins p62, ATG5, and ATG7 in the indicated groups. GAPDH served as a loading control. Densitometric quantification is shown below. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Inhibition, Transfection, Plasmid Preparation, Western Blot, Control, Staining, Expressing, Fluorescence

    Modulation of autophagy and ferroptosis in MM cells through SDE2 manipulation and pharmacological interventions. KMS-11 cells treated with SDE2 knockdown and/or the autophagy inhibitor chloroquine (CQ): (A) Western blot analysis of autophagy-related proteins LC3 and p62 in KMS-11 cells. Densitometric quantification is shown on the right. (B) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells under the same treatments as in (A), with corresponding quantification on the right. (C) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. H929 cells treated with SDE2 overexpression and/or the ferroptosis inducer Erastin: (D) Western blot analysis of autophagy-related proteins LC3 and p62 in H929 cells. Densitometric quantification is shown on the right. (E) Western blot analysis and quantification of ATG5 and ATG7 in H929 cells under the same treatments as in (D). (F) mRFP-GFP-LC3 dual fluorescence labeling visualizing autophagic flux in H929 cells, showing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). KMS-11 cells treated with SDE2 knockdown and/or ATG5 knockdown: (G) Western blot analysis and quantification of Beclin1 and p62 in KMS-11 cells expressing shCtrl or shSDE2, with or without ATG5 knockdown. (H) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells with single or combined knockdown of SDE2 and ATG5. Quantification is shown on the right. (I) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Modulation of autophagy and ferroptosis in MM cells through SDE2 manipulation and pharmacological interventions. KMS-11 cells treated with SDE2 knockdown and/or the autophagy inhibitor chloroquine (CQ): (A) Western blot analysis of autophagy-related proteins LC3 and p62 in KMS-11 cells. Densitometric quantification is shown on the right. (B) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells under the same treatments as in (A), with corresponding quantification on the right. (C) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. H929 cells treated with SDE2 overexpression and/or the ferroptosis inducer Erastin: (D) Western blot analysis of autophagy-related proteins LC3 and p62 in H929 cells. Densitometric quantification is shown on the right. (E) Western blot analysis and quantification of ATG5 and ATG7 in H929 cells under the same treatments as in (D). (F) mRFP-GFP-LC3 dual fluorescence labeling visualizing autophagic flux in H929 cells, showing autophagosomes (yellow puncta: red + green) and autolysosomes (red-only puncta). KMS-11 cells treated with SDE2 knockdown and/or ATG5 knockdown: (G) Western blot analysis and quantification of Beclin1 and p62 in KMS-11 cells expressing shCtrl or shSDE2, with or without ATG5 knockdown. (H) Western blot analysis of ACSL4, FTH1, and GPX4 in KMS-11 cells with single or combined knockdown of SDE2 and ATG5. Quantification is shown on the right. (I) Biochemical analysis of ferroptosis-related markers GSH, MDA, and Fe 2+ in KMS-11 cells. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Knockdown, Western Blot, Over Expression, Fluorescence, Labeling, Expressing

    Functional interplay between ATG5 and SDE2 in MM progression and ferroptosis regulation. (A) Western blot analysis of ATG5 expression in malignant cells (T) and non-malignant bone marrow cells (N) isolated from the peripheral blood of MM patients. (B) Western blot analysis confirming the efficiency of ATG5 overexpression in OPM-2 and H929 cells transfected with a plasmid encoding the full-length ATG5 sequence. (C) Crystal violet staining of Transwell migration assays comparing the migratory ability of OPM-2 and H929 cells with or without ATG5 overexpression. (D) Experimental setup for H929 cells treated with SDE2 overexpression, ATG5 overexpression, or a combination of both. (E) Western blot analysis of SDE2 and ATG5 protein levels in H929 cells from the four groups defined in (D). GAPDH was used as a loading control. (F) (Top) Colony formation assay evaluating the proliferative capacity of H929 cells under different treatments. (Bottom) Transwell assay assessing the migratory ability of H929 cells under different treatments. (G) Biochemical analysis of ferroptosis-related markers (MDA, GSH, and Fe 2+ ) in H929 cells subjected to different treatments. (H) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in H929 cells under different treatments. (I) Western blot analysis showing the effects of different treatments on LC3 expression and p62 degradation in H929 cells. (J) Representative images of tumors formed 35 days after subcutaneous inoculation of differently treated H929 stable cell lines into nude mice. (K) Tumor growth curves showing volume changes of tumors derived from differently treated H929 cell lines over time. (L) Average tumor weights of tumors derived from differently treated H929 cell lines. (M) Western blot analysis of NCOA4, ACSL4, FTH1, and GPX4 expression levels in tumors derived from differently treated H929 cell lines. (N) Western blot analysis showing the activation levels of the ATG5/ATG7 pathway and p62 degradation in tumors derived from differently treated H929 cell lines. (O) Representative images of H&E staining and Ki67 immunohistochemical staining of tumor sections derived from differently treated H929 cell lines. The bar graph quantifies the percentage of proliferating cells (Ki67-positive). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Journal: Redox Biology

    Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

    doi: 10.1016/j.redox.2026.104007

    Figure Lengend Snippet: Functional interplay between ATG5 and SDE2 in MM progression and ferroptosis regulation. (A) Western blot analysis of ATG5 expression in malignant cells (T) and non-malignant bone marrow cells (N) isolated from the peripheral blood of MM patients. (B) Western blot analysis confirming the efficiency of ATG5 overexpression in OPM-2 and H929 cells transfected with a plasmid encoding the full-length ATG5 sequence. (C) Crystal violet staining of Transwell migration assays comparing the migratory ability of OPM-2 and H929 cells with or without ATG5 overexpression. (D) Experimental setup for H929 cells treated with SDE2 overexpression, ATG5 overexpression, or a combination of both. (E) Western blot analysis of SDE2 and ATG5 protein levels in H929 cells from the four groups defined in (D). GAPDH was used as a loading control. (F) (Top) Colony formation assay evaluating the proliferative capacity of H929 cells under different treatments. (Bottom) Transwell assay assessing the migratory ability of H929 cells under different treatments. (G) Biochemical analysis of ferroptosis-related markers (MDA, GSH, and Fe 2+ ) in H929 cells subjected to different treatments. (H) Western blot analysis of ferroptosis-related proteins (NCOA4, ACSL4, FTH1, and GPX4) in H929 cells under different treatments. (I) Western blot analysis showing the effects of different treatments on LC3 expression and p62 degradation in H929 cells. (J) Representative images of tumors formed 35 days after subcutaneous inoculation of differently treated H929 stable cell lines into nude mice. (K) Tumor growth curves showing volume changes of tumors derived from differently treated H929 cell lines over time. (L) Average tumor weights of tumors derived from differently treated H929 cell lines. (M) Western blot analysis of NCOA4, ACSL4, FTH1, and GPX4 expression levels in tumors derived from differently treated H929 cell lines. (N) Western blot analysis showing the activation levels of the ATG5/ATG7 pathway and p62 degradation in tumors derived from differently treated H929 cell lines. (O) Representative images of H&E staining and Ki67 immunohistochemical staining of tumor sections derived from differently treated H929 cell lines. The bar graph quantifies the percentage of proliferating cells (Ki67-positive). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

    Article Snippet: Lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with antibodies against SDE2 (PA5-46779, Thermo Fisher), ATG5 (#2630, Cell Signaling Technology), LC3 (sc-271625, Santa Cruz), P62 (sc-28359, Santa Cruz), and GAPDH (ab8245, Abcam).

    Techniques: Functional Assay, Western Blot, Expressing, Isolation, Over Expression, Transfection, Plasmid Preparation, Sequencing, Staining, Migration, Control, Colony Assay, Transwell Assay, Stable Transfection, Derivative Assay, Activation Assay, Immunohistochemical staining