Structured Review

Bethyl pla
Pla, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla - by Bioz Stars, 2023-11
86/100 stars

Images


Structured Review

Bethyl pla assay
Pla Assay, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla assay/product/Bethyl
Average 95 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla assay - by Bioz Stars, 2023-11
95/100 stars

Images


Structured Review

Bethyl pla
Pla, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla - by Bioz Stars, 2023-11
86/100 stars

Images


Structured Review

Bethyl pla
Pla, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla - by Bioz Stars, 2023-11
86/100 stars

Images


Structured Review

Bethyl pla plus
The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of <t>PLA</t> foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where <t>indicated,</t> <t>antibodies</t> were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.
Pla Plus, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla plus/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla plus - by Bioz Stars, 2023-11
86/100 stars

Images

1) Product Images from "Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks"

Article Title: Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2022.102268

The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of PLA foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where indicated, antibodies were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.
Figure Legend Snippet: The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of PLA foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where indicated, antibodies were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.

Techniques Used: Binding Assay, Sequencing, Transfection, Western Blot, Two Tailed Test, MANN-WHITNEY, Proximity Ligation Assay, Purification, Expressing, Dot Blot, Magnetic Beads, Incubation, Labeling, Negative Control


Structured Review

Bethyl pla plus
(A) Schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. (B) U2OS cells transfected with either Flag-SDE2 Ct WT or ΔSAP (Δ395-451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (*) indicates non-specific bands. (C) Quantification of (B) from more than three independent experiments; error bar: SEM, *** p <0.001, two-tailed Mann-Whitney test. ns, not significant. (D) U2OS cells were transfected with either Flag-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. Scale bar: 10 μm (E) The average number of <t>PLA</t> foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar: mean, **** p <0.0001, two-tailed Mann-Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. (F) Proteins purified from U2OS cells transiently expressing Flag-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo and streptavidin magnetic beads, and analyzed by Western blotting. (G) Quantification of (F) from four independent experiments; error bar: SEM, ** p <0.01, two-way ANOVA. (H) Electrophoretic mobility shift assay (EMSA) of purified GST-SDE2-Flag WT or ΔSAP (Δ395-427) incubated with 6-carboxyfluorescein (6-FAM)-labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 5 nM, protein: 200 nM. NC = negative control, no protein. (I) Quantification of (H) from four independent experiments; error bar: SEM, ** p <0.01, *** p <0.001, two-way ANOVA. (J) EMSA of purified GST-SDE2-Flag incubated with 60-mer ssDNA. Where <t>indicated,</t> <t>antibodies</t> were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50-200 nM.
Pla Plus, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla plus/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla plus - by Bioz Stars, 2023-11
86/100 stars

Images

1) Product Images from "Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks"

Article Title: Extended DNA binding interface beyond the canonical SAP domain contributes to SDE2 function at DNA replication forks

Journal: bioRxiv

doi: 10.1101/2022.05.09.490802

(A) Schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. (B) U2OS cells transfected with either Flag-SDE2 Ct WT or ΔSAP (Δ395-451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (*) indicates non-specific bands. (C) Quantification of (B) from more than three independent experiments; error bar: SEM, *** p <0.001, two-tailed Mann-Whitney test. ns, not significant. (D) U2OS cells were transfected with either Flag-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. Scale bar: 10 μm (E) The average number of PLA foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar: mean, **** p <0.0001, two-tailed Mann-Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. (F) Proteins purified from U2OS cells transiently expressing Flag-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo and streptavidin magnetic beads, and analyzed by Western blotting. (G) Quantification of (F) from four independent experiments; error bar: SEM, ** p <0.01, two-way ANOVA. (H) Electrophoretic mobility shift assay (EMSA) of purified GST-SDE2-Flag WT or ΔSAP (Δ395-427) incubated with 6-carboxyfluorescein (6-FAM)-labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 5 nM, protein: 200 nM. NC = negative control, no protein. (I) Quantification of (H) from four independent experiments; error bar: SEM, ** p <0.01, *** p <0.001, two-way ANOVA. (J) EMSA of purified GST-SDE2-Flag incubated with 60-mer ssDNA. Where indicated, antibodies were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50-200 nM.
Figure Legend Snippet: (A) Schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. (B) U2OS cells transfected with either Flag-SDE2 Ct WT or ΔSAP (Δ395-451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (*) indicates non-specific bands. (C) Quantification of (B) from more than three independent experiments; error bar: SEM, *** p <0.001, two-tailed Mann-Whitney test. ns, not significant. (D) U2OS cells were transfected with either Flag-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. Scale bar: 10 μm (E) The average number of PLA foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar: mean, **** p <0.0001, two-tailed Mann-Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. (F) Proteins purified from U2OS cells transiently expressing Flag-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo and streptavidin magnetic beads, and analyzed by Western blotting. (G) Quantification of (F) from four independent experiments; error bar: SEM, ** p <0.01, two-way ANOVA. (H) Electrophoretic mobility shift assay (EMSA) of purified GST-SDE2-Flag WT or ΔSAP (Δ395-427) incubated with 6-carboxyfluorescein (6-FAM)-labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 5 nM, protein: 200 nM. NC = negative control, no protein. (I) Quantification of (H) from four independent experiments; error bar: SEM, ** p <0.01, *** p <0.001, two-way ANOVA. (J) EMSA of purified GST-SDE2-Flag incubated with 60-mer ssDNA. Where indicated, antibodies were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50-200 nM.

Techniques Used: Sequencing, Transfection, Western Blot, Two Tailed Test, MANN-WHITNEY, Proximity Ligation Assay, Purification, Expressing, Magnetic Beads, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Negative Control


Structured Review

Bethyl pla assay
Pla Assay, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pla assay/product/Bethyl
Average 95 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pla assay - by Bioz Stars, 2023-11
95/100 stars

Images


Structured Review

Bethyl plas
Plas, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plas/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
plas - by Bioz Stars, 2023-11
86/100 stars

Images


Structured Review

Bethyl plas
Plas, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plas/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
plas - by Bioz Stars, 2023-11
86/100 stars

Images


Structured Review

Bethyl plas
Plas, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plas/product/Bethyl
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
plas - by Bioz Stars, 2023-11
86/100 stars

Images

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • pla  (Bethyl)
    86
    Bethyl pla
    Pla, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pla/product/Bethyl
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pla - by Bioz Stars, 2023-11
    86/100 stars
      Buy from Supplier

    95
    Bethyl pla assay
    Pla Assay, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pla assay/product/Bethyl
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pla assay - by Bioz Stars, 2023-11
    95/100 stars
      Buy from Supplier

    86
    Bethyl pla plus
    The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of <t>PLA</t> foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where <t>indicated,</t> <t>antibodies</t> were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.
    Pla Plus, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pla plus/product/Bethyl
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pla plus - by Bioz Stars, 2023-11
    86/100 stars
      Buy from Supplier

    plas  (Bethyl)
    86
    Bethyl plas
    The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of <t>PLA</t> foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where <t>indicated,</t> <t>antibodies</t> were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.
    Plas, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plas/product/Bethyl
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plas - by Bioz Stars, 2023-11
    86/100 stars
      Buy from Supplier

    Image Search Results


    The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of PLA foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where indicated, antibodies were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.

    Journal: The Journal of Biological Chemistry

    Article Title: Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks

    doi: 10.1016/j.jbc.2022.102268

    Figure Lengend Snippet: The SAP domain of SDE2 is required for DNA binding. A , schematic of SDE2 domain structures and sequence alignment of SDE2 SAP with various SAP-domain containing proteins. B , U2OS cells transfected with either FLAG-SDE2 Ct WT or ΔSAP (Δ395–451) were fractionated to separate cytosolic and chromatin-associated protein pools and analyzed by Western blotting. S indicates cytosolic proteins, P2 indicates acid-soluble chromatin-bound proteins. Asterisk (∗) indicates nonspecific bands. C , quantification of ( B ) from more than three independent experiments; error bar represents SD, ∗∗∗ p < 0.001, two-tailed Mann–Whitney test. D , U2OS cells were transfected with either FLAG-SDE2 WT or ΔSAP, and proximity to the replication fork was analyzed using the SIRF proximity ligation assay. The scale bar represents 10 μm. E , the average number of PLA foci per cell. Quantification of three independent experiments (>400 cells per condition); red bar represents mean, ∗∗∗∗ p < 0.0001, two-tailed Mann–Whitney test. Percentage underneath graph indicates percent of total cells positive of PLA signal. F , proteins purified from U2OS cells transiently expressing FLAG-SDE2 WT or ΔSAP were pulled down with biotinylated 80-mer ssDNA oligo (equal input shown in a slot blot and antibiotin immunoblot) and streptavidin magnetic beads and analyzed by Western blotting. G , quantification of ( F ) from four independent experiments; error bar represents SD, ∗∗ p < 0.01, two-way ANOVA. H , EMSA of purified GST-SDE2-FLAG WT or ΔSAP (Δ395–427) incubated with 6-carboxyfluorescein (6-FAM)–labeled 60-mer ssDNA, dsDNA, or splayed-arm DNA. DNA: 50 nM, protein: 200 nM. NC, negative control, no protein. I , quantification of ( H ) from four independent experiments; error bar represents SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA. J , EMSA of purified GST-SDE2-FLAG incubated with 60-mer ssDNA. Where indicated, antibodies were added at a 1:2 ratio for a supershift EMSA. DNA: 50 nM, protein: 50 to 200 nM. GST, glutathione- S -transferase; ns, not significant; PLA, proximity ligation assay; SAP, SAF-A/B, Acinus, PIAS; SDE2, silencing-defective 2.

    Article Snippet: In brief, coverslips were blocked with 40 μl of blocking solution and incubated for 1 h, incubated in 25 μl of the primary antibodies for 1 h (rabbit anti-TIM [Bethyl; catalog no.: A300-961A, 1:500 dilution]; rabbit anti-Biotin [Bethyl; catalog no.: A150-109A, 1:3000 dilution]; mouse anti-Biotin [Jackson ImmunoResearch; catalog no.: 200-002-211, 1:2000 dilution), incubated with the PLA plus and minus probes (secondary antibodies) for 1 h, incubated with the ligase for 30 min, incubated with the rolling polymerase for 1 h at 40 min, and mounted using the provided in situ wet mounting medium, containing 4′,6-diamidino-2-phenylindole.

    Techniques: Binding Assay, Sequencing, Transfection, Western Blot, Two Tailed Test, MANN-WHITNEY, Proximity Ligation Assay, Purification, Expressing, Dot Blot, Magnetic Beads, Incubation, Labeling, Negative Control