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sample buffer  (Boster Bio)


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    Structured Review

    Boster Bio sample buffer
    Sample Buffer, supplied by Boster Bio, used in various techniques. Bioz Stars score: 95/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sample+loading+buffer/SDS-PAGE+Protein+Loading+Buffer/pmc13618977-238-6-8
    Average 95 stars, based on 250 article reviews
    sample buffer - by Bioz Stars, 2026-10
    95/100 stars

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    Related Articles

    Protease Inhibitor:

    Article Title: Fine-tuning ERK activity enables proliferation-differentiation balance during lineage specification of human embryonic stem cells.
    Article Snippet: Coverslips were mounted onto microscope slides, and images were acquired using Nikon Ti2 microscope. .. Cell lysates in RIPA buffer with protease inhibitor cocktail or co-immunoprecipitated protein (Co-IP) samples were mixed with sample loading buffer (Bosterbio, AR1112-10) and boiled for 5 min at 95 °C to denature the proteins. .. It was then kept on ice before resolving using 10%–12.5% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, 3010040001) using a wet tank transfer system.

    Article Title: Fine-tuning ERK activity enables proliferation-differentiation balance during lineage specification of human embryonic stem cells
    Article Snippet: Coverslips were mounted onto microscope slides, and images were acquired using Nikon Ti2 microscope. .. Cell lysates in RIPA buffer with protease inhibitor cocktail or co-immunoprecipitated protein (Co-IP) samples were mixed with sample loading buffer (Bosterbio, AR1112-10) and boiled for 5 min at 95 °C to denature the proteins. .. It was then kept on ice before resolving using 10%–12.5% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, 3010040001) using a wet tank transfer system.

    Gel Electrophoresis:

    Article Title: Copper depletion inhibits electron transport chain activity and nucleotide synthesis to boost leukemia therapy
    Article Snippet: .. Protein samples were prepared in 5X sample buffer (Boster Bio, AR1112) so that 20 μg of protein lysate per sample could be used for SDS–polyacrylamide gel electrophoresis. .. Samples were separated on pre-cast 4–12% or 10–20% gels (Invitrogen XP10205BOX or XP04125BOX) and transferred onto 0.45 uM PVDF (Genesee 83–636R).

    Magnetic Beads:

    Article Title: Hepatic GDP-fucose transporter SLC35C1 attenuates cholestatic liver injury and inflammation by inducing CEACAM1 N153 fucosylation
    Article Snippet: .. The precipitated proteins in magnetic beads were denatured in 2× SDS-PAGE protein loading buffer (BOSTER Biological Technology, AR0131-20) at 100 °C for 10 minutes. ..

    SDS Page:

    Article Title: Hepatic GDP-fucose transporter SLC35C1 attenuates cholestatic liver injury and inflammation by inducing CEACAM1 N153 fucosylation
    Article Snippet: .. The precipitated proteins in magnetic beads were denatured in 2× SDS-PAGE protein loading buffer (BOSTER Biological Technology, AR0131-20) at 100 °C for 10 minutes. ..

    Article Title: Taurine Restores Oocyte Quality by Enhancing Mitochondrial Function in Mice Exposed to Dibutyl Phthalate during Adolescence.
    Article Snippet: Adolescence represents a vulnerable window for ovarian development, during 17 which oocytes rely heavily on mitochondrial bioenergetics and redox homeostasis.. Dibutyl 18 phthalate (DBP) is a widely used plasticizer recognized for its endocrine-disrupting 19 properties.. It can compromise oocyte integrity during these sensitive developmental stages.

    Saline:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 155506; RRID: AB_2750234 BV421 anti-MerTK BioLegend cat. no. 151510; RRID: AB_2832533 APC/Fire-750 anti–major histocompatibility complex (MHC) II BioLegend cat. no. 107652; RRID: AB_2616728 APC anti-CD209b Invitrogen cat. no. 17-2093-82; RRID: AB_11151692 BUV395 anti-CD24 BD Horizon cat. no. 566221; RRID: AB_2632389 BUV805 anti-CD103 BD Horizon cat. no. 741948; RRID: AB_2871259 PE-Cy7 anti-CD32b BioLegend cat. no. 303213; RRID: AB_2616922 PEDazzle 594 anti-CD64 BioLegend cat. no. 305031; RRID: AB_2564186 BUV615 anti-CD16 BD Horizon cat. no. 751572; RRID: AB_2875567 Human anti-kappa Southern Biotech cat. no. 9230-01; RRID: AB_2796705 Human anti-lambda Southern Biotech cat. no. 9180-01; RRID: AB_2796674 HRP–conjugated anti-human IgG secondary antibody Southern Biotech cat. no. 2040-05: RRID: AB_2795644 PerCP-Cy5.5 anti-CD3 BioLegend cat. no. 100218; RRID: AB_1595492 PerCP-Cy5.5 anti-B220 BioLegend cat. no. 103236; RRID: AB_893354 APC-Fire750 anti-NK-1.1 BioLegend cat. no. 156516; RRID: AB_2892323 PE anti-CD11b BioLegend cat. no. 101207; RRID: AB_312790 APC anti-Ly6C BioLegend cat. no. 128016; RRID: AB_1732076 APC-Fire750 anti-Ly6C BioLegend cat. no. 128046; RRID: AB_2616730 BV785 anti-Ly6G BioLegend cat. no. 127645; RRID: AB_2566317 PE anti-myeloid-epithelial-reproductive tyrosine kinase (MerTK) BioLegend cat. no. 151506; RRID: AB_2617036 BV650 anti-human CD16 BioLegend cat. no. 302042; RRID: AB_2563801 APC/Fire 750 anti–I-A/I-E BioLegend cat. no. 107652; RRID: AB_2616728 BV785 anti–I-A/I-E BioLegend cat. no. 107645; RRID: AB_2565977 BV421 anti-CD16 BioLegend cat. no. 302038; RRID: AB_10898112 FITC anti-CD32a STEMCELL cat. no. 60012FI.1 APC anti-CD32b BioLegend cat. no. 398304; RRID: AB_2860997 PerCP-Cy5.5 anti-CD209 BioLegend cat. no. 330110; RRID: AB_1236468 rabbit anti-REST Invitrogen cat. no. MA5-44189; RRID: AB_2926319 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Phosphate buffered saline Gibco cat. no. 14190-144 Non-fat Milk Lab Scientific bioKEMIX cat. no. M0841 SDS sample buffer Boster cat. no. AR1112 Single-cell lysis buffer Takara cat. no. 635013 RNase inhibitor Takara cat. no. 2313B Fixation Buffer BioLegend cat. no. 420801 DharmaFect3 Transfection Reagent Horizon Discovery cat. no. NC1650394 Non-essential amino acids Gibco cat. no. 11-140-050 Sodium pyruvate Gibco cat. no. 11-360-070 Macrophage-colony stimulating factor (M-CSF) Peprotech cat. no. 300-25 IL-4 Peprotech cat. no. 200-04 glycine Sigma-Aldrich cat. no. G8898 TritonX-100 Sigma-Aldrich cat. no. 93443 ProLong Gold Antifade Reagent Cell Signaling Technology cat. no. P36930 Normocin Invivogen cat. no. ant-nr-1 Blasticidin Invivogen cat. no. ant-bl-1 phorbol 12-myristate 13-acetate (PMA) Thermo Fisher Scientific cat. no. J63916.MCR lipopolysaccharide (LPS) eBioscience cat. no. 50-112-2025 QUANTI-Blue solution Invivogen cat. no. rep-qbs Hoechst 33342 Abcam Inc. cat. no. ab228551 Alexa Fluor 488 phalloidin Invitrogen cat. no. A12379 LIVE/DEAD Blue Fixable Dye Thermo Fisher Scientific cat. no. L34962 LIVE/DEAD Aqua Fixable Dye Thermo Fisher Scientific cat. no. L34957 Critical Commercial Assays Nano-Glo Luciferase Assay System Promega cat. no. N1120 Legendplex Bead array kits Biolegend cat. nos. .. 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    Milk:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 155506; RRID: AB_2750234 BV421 anti-MerTK BioLegend cat. no. 151510; RRID: AB_2832533 APC/Fire-750 anti–major histocompatibility complex (MHC) II BioLegend cat. no. 107652; RRID: AB_2616728 APC anti-CD209b Invitrogen cat. no. 17-2093-82; RRID: AB_11151692 BUV395 anti-CD24 BD Horizon cat. no. 566221; RRID: AB_2632389 BUV805 anti-CD103 BD Horizon cat. no. 741948; RRID: AB_2871259 PE-Cy7 anti-CD32b BioLegend cat. no. 303213; RRID: AB_2616922 PEDazzle 594 anti-CD64 BioLegend cat. no. 305031; RRID: AB_2564186 BUV615 anti-CD16 BD Horizon cat. no. 751572; RRID: AB_2875567 Human anti-kappa Southern Biotech cat. no. 9230-01; RRID: AB_2796705 Human anti-lambda Southern Biotech cat. no. 9180-01; RRID: AB_2796674 HRP–conjugated anti-human IgG secondary antibody Southern Biotech cat. no. 2040-05: RRID: AB_2795644 PerCP-Cy5.5 anti-CD3 BioLegend cat. no. 100218; RRID: AB_1595492 PerCP-Cy5.5 anti-B220 BioLegend cat. no. 103236; RRID: AB_893354 APC-Fire750 anti-NK-1.1 BioLegend cat. no. 156516; RRID: AB_2892323 PE anti-CD11b BioLegend cat. no. 101207; RRID: AB_312790 APC anti-Ly6C BioLegend cat. no. 128016; RRID: AB_1732076 APC-Fire750 anti-Ly6C BioLegend cat. no. 128046; RRID: AB_2616730 BV785 anti-Ly6G BioLegend cat. no. 127645; RRID: AB_2566317 PE anti-myeloid-epithelial-reproductive tyrosine kinase (MerTK) BioLegend cat. no. 151506; RRID: AB_2617036 BV650 anti-human CD16 BioLegend cat. no. 302042; RRID: AB_2563801 APC/Fire 750 anti–I-A/I-E BioLegend cat. no. 107652; RRID: AB_2616728 BV785 anti–I-A/I-E BioLegend cat. no. 107645; RRID: AB_2565977 BV421 anti-CD16 BioLegend cat. no. 302038; RRID: AB_10898112 FITC anti-CD32a STEMCELL cat. no. 60012FI.1 APC anti-CD32b BioLegend cat. no. 398304; RRID: AB_2860997 PerCP-Cy5.5 anti-CD209 BioLegend cat. no. 330110; RRID: AB_1236468 rabbit anti-REST Invitrogen cat. no. MA5-44189; RRID: AB_2926319 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Phosphate buffered saline Gibco cat. no. 14190-144 Non-fat Milk Lab Scientific bioKEMIX cat. no. M0841 SDS sample buffer Boster cat. no. AR1112 Single-cell lysis buffer Takara cat. no. 635013 RNase inhibitor Takara cat. no. 2313B Fixation Buffer BioLegend cat. no. 420801 DharmaFect3 Transfection Reagent Horizon Discovery cat. no. NC1650394 Non-essential amino acids Gibco cat. no. 11-140-050 Sodium pyruvate Gibco cat. no. 11-360-070 Macrophage-colony stimulating factor (M-CSF) Peprotech cat. no. 300-25 IL-4 Peprotech cat. no. 200-04 glycine Sigma-Aldrich cat. no. G8898 TritonX-100 Sigma-Aldrich cat. no. 93443 ProLong Gold Antifade Reagent Cell Signaling Technology cat. no. P36930 Normocin Invivogen cat. no. ant-nr-1 Blasticidin Invivogen cat. no. ant-bl-1 phorbol 12-myristate 13-acetate (PMA) Thermo Fisher Scientific cat. no. J63916.MCR lipopolysaccharide (LPS) eBioscience cat. no. 50-112-2025 QUANTI-Blue solution Invivogen cat. no. rep-qbs Hoechst 33342 Abcam Inc. cat. no. ab228551 Alexa Fluor 488 phalloidin Invitrogen cat. no. A12379 LIVE/DEAD Blue Fixable Dye Thermo Fisher Scientific cat. no. L34962 LIVE/DEAD Aqua Fixable Dye Thermo Fisher Scientific cat. no. L34957 Critical Commercial Assays Nano-Glo Luciferase Assay System Promega cat. no. N1120 Legendplex Bead array kits Biolegend cat. nos. .. 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    Single Cell:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 155506; RRID: AB_2750234 BV421 anti-MerTK BioLegend cat. no. 151510; RRID: AB_2832533 APC/Fire-750 anti–major histocompatibility complex (MHC) II BioLegend cat. no. 107652; RRID: AB_2616728 APC anti-CD209b Invitrogen cat. no. 17-2093-82; RRID: AB_11151692 BUV395 anti-CD24 BD Horizon cat. no. 566221; RRID: AB_2632389 BUV805 anti-CD103 BD Horizon cat. no. 741948; RRID: AB_2871259 PE-Cy7 anti-CD32b BioLegend cat. no. 303213; RRID: AB_2616922 PEDazzle 594 anti-CD64 BioLegend cat. no. 305031; RRID: AB_2564186 BUV615 anti-CD16 BD Horizon cat. no. 751572; RRID: AB_2875567 Human anti-kappa Southern Biotech cat. no. 9230-01; RRID: AB_2796705 Human anti-lambda Southern Biotech cat. no. 9180-01; RRID: AB_2796674 HRP–conjugated anti-human IgG secondary antibody Southern Biotech cat. no. 2040-05: RRID: AB_2795644 PerCP-Cy5.5 anti-CD3 BioLegend cat. no. 100218; RRID: AB_1595492 PerCP-Cy5.5 anti-B220 BioLegend cat. no. 103236; RRID: AB_893354 APC-Fire750 anti-NK-1.1 BioLegend cat. no. 156516; RRID: AB_2892323 PE anti-CD11b BioLegend cat. no. 101207; RRID: AB_312790 APC anti-Ly6C BioLegend cat. no. 128016; RRID: AB_1732076 APC-Fire750 anti-Ly6C BioLegend cat. no. 128046; RRID: AB_2616730 BV785 anti-Ly6G BioLegend cat. no. 127645; RRID: AB_2566317 PE anti-myeloid-epithelial-reproductive tyrosine kinase (MerTK) BioLegend cat. no. 151506; RRID: AB_2617036 BV650 anti-human CD16 BioLegend cat. no. 302042; RRID: AB_2563801 APC/Fire 750 anti–I-A/I-E BioLegend cat. no. 107652; RRID: AB_2616728 BV785 anti–I-A/I-E BioLegend cat. no. 107645; RRID: AB_2565977 BV421 anti-CD16 BioLegend cat. no. 302038; RRID: AB_10898112 FITC anti-CD32a STEMCELL cat. no. 60012FI.1 APC anti-CD32b BioLegend cat. no. 398304; RRID: AB_2860997 PerCP-Cy5.5 anti-CD209 BioLegend cat. no. 330110; RRID: AB_1236468 rabbit anti-REST Invitrogen cat. no. MA5-44189; RRID: AB_2926319 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Phosphate buffered saline Gibco cat. no. 14190-144 Non-fat Milk Lab Scientific bioKEMIX cat. no. M0841 SDS sample buffer Boster cat. no. AR1112 Single-cell lysis buffer Takara cat. no. 635013 RNase inhibitor Takara cat. no. 2313B Fixation Buffer BioLegend cat. no. 420801 DharmaFect3 Transfection Reagent Horizon Discovery cat. no. NC1650394 Non-essential amino acids Gibco cat. no. 11-140-050 Sodium pyruvate Gibco cat. no. 11-360-070 Macrophage-colony stimulating factor (M-CSF) Peprotech cat. no. 300-25 IL-4 Peprotech cat. no. 200-04 glycine Sigma-Aldrich cat. no. G8898 TritonX-100 Sigma-Aldrich cat. no. 93443 ProLong Gold Antifade Reagent Cell Signaling Technology cat. no. P36930 Normocin Invivogen cat. no. ant-nr-1 Blasticidin Invivogen cat. no. ant-bl-1 phorbol 12-myristate 13-acetate (PMA) Thermo Fisher Scientific cat. no. J63916.MCR lipopolysaccharide (LPS) eBioscience cat. no. 50-112-2025 QUANTI-Blue solution Invivogen cat. no. rep-qbs Hoechst 33342 Abcam Inc. cat. no. ab228551 Alexa Fluor 488 phalloidin Invitrogen cat. no. A12379 LIVE/DEAD Blue Fixable Dye Thermo Fisher Scientific cat. no. L34962 LIVE/DEAD Aqua Fixable Dye Thermo Fisher Scientific cat. no. L34957 Critical Commercial Assays Nano-Glo Luciferase Assay System Promega cat. no. N1120 Legendplex Bead array kits Biolegend cat. nos. .. 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    Lysis:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 155506; RRID: AB_2750234 BV421 anti-MerTK BioLegend cat. no. 151510; RRID: AB_2832533 APC/Fire-750 anti–major histocompatibility complex (MHC) II BioLegend cat. no. 107652; RRID: AB_2616728 APC anti-CD209b Invitrogen cat. no. 17-2093-82; RRID: AB_11151692 BUV395 anti-CD24 BD Horizon cat. no. 566221; RRID: AB_2632389 BUV805 anti-CD103 BD Horizon cat. no. 741948; RRID: AB_2871259 PE-Cy7 anti-CD32b BioLegend cat. no. 303213; RRID: AB_2616922 PEDazzle 594 anti-CD64 BioLegend cat. no. 305031; RRID: AB_2564186 BUV615 anti-CD16 BD Horizon cat. no. 751572; RRID: AB_2875567 Human anti-kappa Southern Biotech cat. no. 9230-01; RRID: AB_2796705 Human anti-lambda Southern Biotech cat. no. 9180-01; RRID: AB_2796674 HRP–conjugated anti-human IgG secondary antibody Southern Biotech cat. no. 2040-05: RRID: AB_2795644 PerCP-Cy5.5 anti-CD3 BioLegend cat. no. 100218; RRID: AB_1595492 PerCP-Cy5.5 anti-B220 BioLegend cat. no. 103236; RRID: AB_893354 APC-Fire750 anti-NK-1.1 BioLegend cat. no. 156516; RRID: AB_2892323 PE anti-CD11b BioLegend cat. no. 101207; RRID: AB_312790 APC anti-Ly6C BioLegend cat. no. 128016; RRID: AB_1732076 APC-Fire750 anti-Ly6C BioLegend cat. no. 128046; RRID: AB_2616730 BV785 anti-Ly6G BioLegend cat. no. 127645; RRID: AB_2566317 PE anti-myeloid-epithelial-reproductive tyrosine kinase (MerTK) BioLegend cat. no. 151506; RRID: AB_2617036 BV650 anti-human CD16 BioLegend cat. no. 302042; RRID: AB_2563801 APC/Fire 750 anti–I-A/I-E BioLegend cat. no. 107652; RRID: AB_2616728 BV785 anti–I-A/I-E BioLegend cat. no. 107645; RRID: AB_2565977 BV421 anti-CD16 BioLegend cat. no. 302038; RRID: AB_10898112 FITC anti-CD32a STEMCELL cat. no. 60012FI.1 APC anti-CD32b BioLegend cat. no. 398304; RRID: AB_2860997 PerCP-Cy5.5 anti-CD209 BioLegend cat. no. 330110; RRID: AB_1236468 rabbit anti-REST Invitrogen cat. no. MA5-44189; RRID: AB_2926319 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Phosphate buffered saline Gibco cat. no. 14190-144 Non-fat Milk Lab Scientific bioKEMIX cat. no. M0841 SDS sample buffer Boster cat. no. AR1112 Single-cell lysis buffer Takara cat. no. 635013 RNase inhibitor Takara cat. no. 2313B Fixation Buffer BioLegend cat. no. 420801 DharmaFect3 Transfection Reagent Horizon Discovery cat. no. NC1650394 Non-essential amino acids Gibco cat. no. 11-140-050 Sodium pyruvate Gibco cat. no. 11-360-070 Macrophage-colony stimulating factor (M-CSF) Peprotech cat. no. 300-25 IL-4 Peprotech cat. no. 200-04 glycine Sigma-Aldrich cat. no. G8898 TritonX-100 Sigma-Aldrich cat. no. 93443 ProLong Gold Antifade Reagent Cell Signaling Technology cat. no. P36930 Normocin Invivogen cat. no. ant-nr-1 Blasticidin Invivogen cat. no. ant-bl-1 phorbol 12-myristate 13-acetate (PMA) Thermo Fisher Scientific cat. no. J63916.MCR lipopolysaccharide (LPS) eBioscience cat. no. 50-112-2025 QUANTI-Blue solution Invivogen cat. no. rep-qbs Hoechst 33342 Abcam Inc. cat. no. ab228551 Alexa Fluor 488 phalloidin Invitrogen cat. no. A12379 LIVE/DEAD Blue Fixable Dye Thermo Fisher Scientific cat. no. L34962 LIVE/DEAD Aqua Fixable Dye Thermo Fisher Scientific cat. no. L34957 Critical Commercial Assays Nano-Glo Luciferase Assay System Promega cat. no. N1120 Legendplex Bead array kits Biolegend cat. nos. .. 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    Transfection:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 155506; RRID: AB_2750234 BV421 anti-MerTK BioLegend cat. no. 151510; RRID: AB_2832533 APC/Fire-750 anti–major histocompatibility complex (MHC) II BioLegend cat. no. 107652; RRID: AB_2616728 APC anti-CD209b Invitrogen cat. no. 17-2093-82; RRID: AB_11151692 BUV395 anti-CD24 BD Horizon cat. no. 566221; RRID: AB_2632389 BUV805 anti-CD103 BD Horizon cat. no. 741948; RRID: AB_2871259 PE-Cy7 anti-CD32b BioLegend cat. no. 303213; RRID: AB_2616922 PEDazzle 594 anti-CD64 BioLegend cat. no. 305031; RRID: AB_2564186 BUV615 anti-CD16 BD Horizon cat. no. 751572; RRID: AB_2875567 Human anti-kappa Southern Biotech cat. no. 9230-01; RRID: AB_2796705 Human anti-lambda Southern Biotech cat. no. 9180-01; RRID: AB_2796674 HRP–conjugated anti-human IgG secondary antibody Southern Biotech cat. no. 2040-05: RRID: AB_2795644 PerCP-Cy5.5 anti-CD3 BioLegend cat. no. 100218; RRID: AB_1595492 PerCP-Cy5.5 anti-B220 BioLegend cat. no. 103236; RRID: AB_893354 APC-Fire750 anti-NK-1.1 BioLegend cat. no. 156516; RRID: AB_2892323 PE anti-CD11b BioLegend cat. no. 101207; RRID: AB_312790 APC anti-Ly6C BioLegend cat. no. 128016; RRID: AB_1732076 APC-Fire750 anti-Ly6C BioLegend cat. no. 128046; RRID: AB_2616730 BV785 anti-Ly6G BioLegend cat. no. 127645; RRID: AB_2566317 PE anti-myeloid-epithelial-reproductive tyrosine kinase (MerTK) BioLegend cat. no. 151506; RRID: AB_2617036 BV650 anti-human CD16 BioLegend cat. no. 302042; RRID: AB_2563801 APC/Fire 750 anti–I-A/I-E BioLegend cat. no. 107652; RRID: AB_2616728 BV785 anti–I-A/I-E BioLegend cat. no. 107645; RRID: AB_2565977 BV421 anti-CD16 BioLegend cat. no. 302038; RRID: AB_10898112 FITC anti-CD32a STEMCELL cat. no. 60012FI.1 APC anti-CD32b BioLegend cat. no. 398304; RRID: AB_2860997 PerCP-Cy5.5 anti-CD209 BioLegend cat. no. 330110; RRID: AB_1236468 rabbit anti-REST Invitrogen cat. no. MA5-44189; RRID: AB_2926319 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Phosphate buffered saline Gibco cat. no. 14190-144 Non-fat Milk Lab Scientific bioKEMIX cat. no. M0841 SDS sample buffer Boster cat. no. AR1112 Single-cell lysis buffer Takara cat. no. 635013 RNase inhibitor Takara cat. no. 2313B Fixation Buffer BioLegend cat. no. 420801 DharmaFect3 Transfection Reagent Horizon Discovery cat. no. NC1650394 Non-essential amino acids Gibco cat. no. 11-140-050 Sodium pyruvate Gibco cat. no. 11-360-070 Macrophage-colony stimulating factor (M-CSF) Peprotech cat. no. 300-25 IL-4 Peprotech cat. no. 200-04 glycine Sigma-Aldrich cat. no. G8898 TritonX-100 Sigma-Aldrich cat. no. 93443 ProLong Gold Antifade Reagent Cell Signaling Technology cat. no. P36930 Normocin Invivogen cat. no. ant-nr-1 Blasticidin Invivogen cat. no. ant-bl-1 phorbol 12-myristate 13-acetate (PMA) Thermo Fisher Scientific cat. no. J63916.MCR lipopolysaccharide (LPS) eBioscience cat. no. 50-112-2025 QUANTI-Blue solution Invivogen cat. no. rep-qbs Hoechst 33342 Abcam Inc. cat. no. ab228551 Alexa Fluor 488 phalloidin Invitrogen cat. no. A12379 LIVE/DEAD Blue Fixable Dye Thermo Fisher Scientific cat. no. L34962 LIVE/DEAD Aqua Fixable Dye Thermo Fisher Scientific cat. no. L34957 Critical Commercial Assays Nano-Glo Luciferase Assay System Promega cat. no. N1120 Legendplex Bead array kits Biolegend cat. nos. .. 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    Luciferase:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 155506; RRID: AB_2750234 BV421 anti-MerTK BioLegend cat. no. 151510; RRID: AB_2832533 APC/Fire-750 anti–major histocompatibility complex (MHC) II BioLegend cat. no. 107652; RRID: AB_2616728 APC anti-CD209b Invitrogen cat. no. 17-2093-82; RRID: AB_11151692 BUV395 anti-CD24 BD Horizon cat. no. 566221; RRID: AB_2632389 BUV805 anti-CD103 BD Horizon cat. no. 741948; RRID: AB_2871259 PE-Cy7 anti-CD32b BioLegend cat. no. 303213; RRID: AB_2616922 PEDazzle 594 anti-CD64 BioLegend cat. no. 305031; RRID: AB_2564186 BUV615 anti-CD16 BD Horizon cat. no. 751572; RRID: AB_2875567 Human anti-kappa Southern Biotech cat. no. 9230-01; RRID: AB_2796705 Human anti-lambda Southern Biotech cat. no. 9180-01; RRID: AB_2796674 HRP–conjugated anti-human IgG secondary antibody Southern Biotech cat. no. 2040-05: RRID: AB_2795644 PerCP-Cy5.5 anti-CD3 BioLegend cat. no. 100218; RRID: AB_1595492 PerCP-Cy5.5 anti-B220 BioLegend cat. no. 103236; RRID: AB_893354 APC-Fire750 anti-NK-1.1 BioLegend cat. no. 156516; RRID: AB_2892323 PE anti-CD11b BioLegend cat. no. 101207; RRID: AB_312790 APC anti-Ly6C BioLegend cat. no. 128016; RRID: AB_1732076 APC-Fire750 anti-Ly6C BioLegend cat. no. 128046; RRID: AB_2616730 BV785 anti-Ly6G BioLegend cat. no. 127645; RRID: AB_2566317 PE anti-myeloid-epithelial-reproductive tyrosine kinase (MerTK) BioLegend cat. no. 151506; RRID: AB_2617036 BV650 anti-human CD16 BioLegend cat. no. 302042; RRID: AB_2563801 APC/Fire 750 anti–I-A/I-E BioLegend cat. no. 107652; RRID: AB_2616728 BV785 anti–I-A/I-E BioLegend cat. no. 107645; RRID: AB_2565977 BV421 anti-CD16 BioLegend cat. no. 302038; RRID: AB_10898112 FITC anti-CD32a STEMCELL cat. no. 60012FI.1 APC anti-CD32b BioLegend cat. no. 398304; RRID: AB_2860997 PerCP-Cy5.5 anti-CD209 BioLegend cat. no. 330110; RRID: AB_1236468 rabbit anti-REST Invitrogen cat. no. MA5-44189; RRID: AB_2926319 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Phosphate buffered saline Gibco cat. no. 14190-144 Non-fat Milk Lab Scientific bioKEMIX cat. no. M0841 SDS sample buffer Boster cat. no. AR1112 Single-cell lysis buffer Takara cat. no. 635013 RNase inhibitor Takara cat. no. 2313B Fixation Buffer BioLegend cat. no. 420801 DharmaFect3 Transfection Reagent Horizon Discovery cat. no. NC1650394 Non-essential amino acids Gibco cat. no. 11-140-050 Sodium pyruvate Gibco cat. no. 11-360-070 Macrophage-colony stimulating factor (M-CSF) Peprotech cat. no. 300-25 IL-4 Peprotech cat. no. 200-04 glycine Sigma-Aldrich cat. no. G8898 TritonX-100 Sigma-Aldrich cat. no. 93443 ProLong Gold Antifade Reagent Cell Signaling Technology cat. no. P36930 Normocin Invivogen cat. no. ant-nr-1 Blasticidin Invivogen cat. no. ant-bl-1 phorbol 12-myristate 13-acetate (PMA) Thermo Fisher Scientific cat. no. J63916.MCR lipopolysaccharide (LPS) eBioscience cat. no. 50-112-2025 QUANTI-Blue solution Invivogen cat. no. rep-qbs Hoechst 33342 Abcam Inc. cat. no. ab228551 Alexa Fluor 488 phalloidin Invitrogen cat. no. A12379 LIVE/DEAD Blue Fixable Dye Thermo Fisher Scientific cat. no. L34962 LIVE/DEAD Aqua Fixable Dye Thermo Fisher Scientific cat. no. L34957 Critical Commercial Assays Nano-Glo Luciferase Assay System Promega cat. no. N1120 Legendplex Bead array kits Biolegend cat. nos. .. 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    other:

    Article Title: Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease.
    Article Snippet: 740390, 740451 RNeasy Micro Kit Qiagen cat. no. 74004 Gibson Assembly New England BioLabs cat. no. E2611L EasySep PE Positive Selection Kit II STEMCELL cat. no. 17684 SMART-Seq v4 Ultra Low Input RNA kit Takara cat. no. 634888 Nextera XT library preparation kit Illumina cat. no. FC-131-1096 Deposited Data Data from human PBMC of influenza patients from public datasets Liu et al.,55 Zhai et al.,56 Sweeney et al.,57 Wong et al.58–60 Ramilo et al.,61 Parnell et al.,62 Berdal et al.,63 Bermejo-Martin et al.,64 Parnell et al.,65 Zaas et al.,66 Dunning et al.,67 and Tang et al.68 GSE73072, GSE68310, GSE66099, GSE4607, GSE6269, GSE40012, GSE27131, GSE21802, GSE20346, GSE17156, GSE111368, GSE101702 Data for generating figures This paper Immport SDY2810 Experimental models: Cell lines MDCK cell BEI and Dr. Gene Tan N/A FreeStyle 293-F cells Thermo Fisher cat. no. R790-07 THP1-Blue NF-kB cells Invivogen cat. no. thp-nfkbv2 (Continued on next page) Immunity 58, 182–196.e1–e10, January 14, 2025 e3

    Western Blot:

    Article Title: Taurine Restores Oocyte Quality by Enhancing Mitochondrial Function in Mice Exposed to Dibutyl Phthalate during Adolescence.
    Article Snippet: Adolescence represents a vulnerable window for ovarian development, during 17 which oocytes rely heavily on mitochondrial bioenergetics and redox homeostasis.. Dibutyl 18 phthalate (DBP) is a widely used plasticizer recognized for its endocrine-disrupting 19 properties.. It can compromise oocyte integrity during these sensitive developmental stages.

    Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study
    Article Snippet: .. One half of the beads was eluted with SDS loading buffer (Boster) and subsequently boiled at 95 °C for 10 min for western blot analysis, while the other half was eluted with chromatin immunoprecipitation (ChIP) elution buffer (5 mM Tris–HCl, 10 mM EDTA, and 1 % SDS). .. Following proteinase K treatment, DNA extraction was performed using the TIANamp Genomic DNA Kit (TaKaRa), followed by standard PCR analysis as previously described.

    Chromatin Immunoprecipitation:

    Article Title: Humanin reduces nucleus pulposus cells ferroptosis to alleviate intervertebral disc degeneration: An in vitro and in vivo study
    Article Snippet: .. One half of the beads was eluted with SDS loading buffer (Boster) and subsequently boiled at 95 °C for 10 min for western blot analysis, while the other half was eluted with chromatin immunoprecipitation (ChIP) elution buffer (5 mM Tris–HCl, 10 mM EDTA, and 1 % SDS). .. Following proteinase K treatment, DNA extraction was performed using the TIANamp Genomic DNA Kit (TaKaRa), followed by standard PCR analysis as previously described.



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    a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b <t>,</t> <t>SDS-PAGE</t> analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.
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    a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b , SDS-PAGE analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.

    Journal: bioRxiv

    Article Title: Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

    doi: 10.64898/2026.06.05.730395

    Figure Lengend Snippet: a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b , SDS-PAGE analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.

    Article Snippet: Reactions were stopped with the addition of 3 μL 4x SDS-PAGE loading dye [240 mM Tris-HCl pH 6.8, 40% (v/v) glycerol, 8% (w/v) SDS, 0.04% (w/v) bromophenol blue, and 5% (v/v) β-Mercaptoethanol], and products were separated on 4-12% or 12% Nu-PAGE Bis-Tris gels (Invitrogen).

    Techniques: Construct, Ubiquitin Proteomics, Variant Assay, SDS Page, Incubation, Activity Assay, Silver Staining, Fluorescence, Staining, Molecular Weight

    a , K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or the indicated ARISC variants (5 nM) for 60 minutes. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. b, SDS-PAGE analysis of ARISC(E33A)–RAP80, ARISC(E33A) BRCC36(S98K) –RAP80, ARISC(E33A) Abraxas1(Δ42-55) –RAP80, and ARISC(E33A) BRCC45(ΔLoop) –RAP80. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. c, Spectral shift assays measuring binding of labelled ARISC(E33A)–RAP80 or the indicated mutant complexes (40 nM) to cyclical K63-Ub6 chains (20 µM-0 µM). Data points are mean ± SEM of two independent experiments carried out in technical duplicates. Dissociation constants (K d ) are indicated; CI, confidence interval. d, Representative images of WT or mutants BRCC36 IRIF in HT-29 cells 4 h post irradiation (10 Gy). Scale bar is 10 µm. e, Western blots showing BRCC36 protein levels in HT-29 cells reconstituted with WT or mutants BRCC36 as indicated (l eft ). Scatter plot showing quantification of the BRCC36 IRIF described in d . Data represent mean ± SEM derived from n ≥ 300 nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test ( right ). f, K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or ARISC Δ42-55 (Abraxas1 Δ42-55) (5 nM) for up to 60 minutes. Cleavage activity was analysed as in a . Data are representative of two independent experiments. DUB, deubiquitylating enzyme; WT, wild type; Ub, ubiquitin.

    Journal: bioRxiv

    Article Title: Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

    doi: 10.64898/2026.06.05.730395

    Figure Lengend Snippet: a , K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or the indicated ARISC variants (5 nM) for 60 minutes. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. b, SDS-PAGE analysis of ARISC(E33A)–RAP80, ARISC(E33A) BRCC36(S98K) –RAP80, ARISC(E33A) Abraxas1(Δ42-55) –RAP80, and ARISC(E33A) BRCC45(ΔLoop) –RAP80. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. c, Spectral shift assays measuring binding of labelled ARISC(E33A)–RAP80 or the indicated mutant complexes (40 nM) to cyclical K63-Ub6 chains (20 µM-0 µM). Data points are mean ± SEM of two independent experiments carried out in technical duplicates. Dissociation constants (K d ) are indicated; CI, confidence interval. d, Representative images of WT or mutants BRCC36 IRIF in HT-29 cells 4 h post irradiation (10 Gy). Scale bar is 10 µm. e, Western blots showing BRCC36 protein levels in HT-29 cells reconstituted with WT or mutants BRCC36 as indicated (l eft ). Scatter plot showing quantification of the BRCC36 IRIF described in d . Data represent mean ± SEM derived from n ≥ 300 nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test ( right ). f, K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or ARISC Δ42-55 (Abraxas1 Δ42-55) (5 nM) for up to 60 minutes. Cleavage activity was analysed as in a . Data are representative of two independent experiments. DUB, deubiquitylating enzyme; WT, wild type; Ub, ubiquitin.

    Article Snippet: Reactions were stopped with the addition of 3 μL 4x SDS-PAGE loading dye [240 mM Tris-HCl pH 6.8, 40% (v/v) glycerol, 8% (w/v) SDS, 0.04% (w/v) bromophenol blue, and 5% (v/v) β-Mercaptoethanol], and products were separated on 4-12% or 12% Nu-PAGE Bis-Tris gels (Invitrogen).

    Techniques: Incubation, Activity Assay, SDS Page, Silver Staining, Binding Assay, Mutagenesis, Irradiation, Western Blot, Derivative Assay, Two Tailed Test, Ubiquitin Proteomics