Structured Review

Millipore phospho rpa32 s33
a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of <t>pRPA2(S33)</t> (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.
Phospho Rpa32 S33, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Price from $9.99 to $1999.99
phospho rpa32 s33 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "Break induced replication orchestrates resection dependent template switch"

Article Title: Break induced replication orchestrates resection dependent template switch

Journal: Nature

doi: 10.1038/s41586-023-06177-3

a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.
Figure Legend Snippet: a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

Techniques Used: SDS Page, Labeling, Silver Staining, Western Blot, Two Tailed Test


Structured Review

Millipore phospho rpa32 s33
a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of <t>pRPA2(S33)</t> (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.
Phospho Rpa32 S33, supplied by Millipore, 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/phospho rpa32 s33/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
phospho rpa32 s33 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "Break induced replication orchestrates resection dependent template switch"

Article Title: Break induced replication orchestrates resection dependent template switch

Journal: Nature

doi: 10.1038/s41586-023-06177-3

a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.
Figure Legend Snippet: a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

Techniques Used: SDS Page, Labeling, Silver Staining, Western Blot, Two Tailed Test


Structured Review

Millipore rpa32
Rpa32, supplied by Millipore, 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/rpa32/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rpa32 - by Bioz Stars, 2024-04
86/100 stars

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Structured Review

Millipore rpa32
Rpa32, supplied by Millipore, 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/rpa32/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rpa32 - by Bioz Stars, 2024-04
86/100 stars

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Structured Review

Millipore rpa32
Rpa32, supplied by Millipore, 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/rpa32/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rpa32 - by Bioz Stars, 2024-04
86/100 stars

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Structured Review

Millipore rpa32
Rpa32, supplied by Millipore, 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/rpa32/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rpa32 - by Bioz Stars, 2024-04
86/100 stars

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Millipore anti rpa32 conjugated dynabeads
A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and <t>RPA32</t> subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).
Anti Rpa32 Conjugated Dynabeads, supplied by Millipore, 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/anti rpa32 conjugated dynabeads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti rpa32 conjugated dynabeads - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "Phosphorylation-Dependent Association of WRN with RPA is Required for Recovery of Replication Forks Stalled at Secondary DNA Structures"

Article Title: Phosphorylation-Dependent Association of WRN with RPA is Required for Recovery of Replication Forks Stalled at Secondary DNA Structures

Journal: bioRxiv

doi: 10.1101/2023.08.08.552428

A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and RPA32 subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).
Figure Legend Snippet: A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and RPA32 subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).

Techniques Used: Staining, Western Blot, Immunoprecipitation, Transfection, Construct, In Situ, Proximity Ligation Assay


Structured Review

Millipore phospho rpa32 s33
Phospho Rpa32 S33, supplied by Millipore, 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/phospho rpa32 s33/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
phospho rpa32 s33 - by Bioz Stars, 2024-04
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Millipore mouse anti rpa32
Mouse Anti Rpa32, supplied by Millipore, 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/mouse anti rpa32/product/Millipore
Average 86 stars, based on 1 article reviews
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mouse anti rpa32 - by Bioz Stars, 2024-04
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Millipore mouse anti phospho rpa32 rpa2
Mouse Anti Phospho Rpa32 Rpa2, supplied by Millipore, 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/mouse anti phospho rpa32 rpa2/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse anti phospho rpa32 rpa2 - by Bioz Stars, 2024-04
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  • 86
    Millipore phospho rpa32 s33
    a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of <t>pRPA2(S33)</t> (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.
    Phospho Rpa32 S33, supplied by Millipore, 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/phospho rpa32 s33/product/Millipore
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    phospho rpa32 s33 - by Bioz Stars, 2024-04
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    86
    Millipore rpa32
    a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of <t>pRPA2(S33)</t> (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.
    Rpa32, supplied by Millipore, 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/rpa32/product/Millipore
    Average 86 stars, based on 1 article reviews
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    rpa32 - by Bioz Stars, 2024-04
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    86
    Millipore anti rpa32 conjugated dynabeads
    A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and <t>RPA32</t> subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).
    Anti Rpa32 Conjugated Dynabeads, supplied by Millipore, 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/anti rpa32 conjugated dynabeads/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rpa32 conjugated dynabeads - by Bioz Stars, 2024-04
    86/100 stars
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    86
    Millipore mouse anti rpa32
    A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and <t>RPA32</t> subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).
    Mouse Anti Rpa32, supplied by Millipore, 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/mouse anti rpa32/product/Millipore
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    Price from $9.99 to $1999.99
    mouse anti rpa32 - by Bioz Stars, 2024-04
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    86
    Millipore mouse anti phospho rpa32 rpa2
    A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and <t>RPA32</t> subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).
    Mouse Anti Phospho Rpa32 Rpa2, supplied by Millipore, 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/mouse anti phospho rpa32 rpa2/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti phospho rpa32 rpa2 - by Bioz Stars, 2024-04
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    Image Search Results


    a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

    Journal: Nature

    Article Title: Break induced replication orchestrates resection dependent template switch

    doi: 10.1038/s41586-023-06177-3

    Figure Lengend Snippet: a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 hrs. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells induced with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

    Article Snippet: The following primary antibodies were used for immunofluorescence: HA.11 (1:200, 901514, Biolegend), RPA2 (Anti-RPA, clone RPA34–20) (1:200, MABE285, EMD Millipore), Phospho RPA32 (S33) (1:500, A300–246A, Bethyl Laboratories).

    Techniques: SDS Page, Labeling, Silver Staining, Western Blot, Two Tailed Test

    A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and RPA32 subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).

    Journal: bioRxiv

    Article Title: Phosphorylation-Dependent Association of WRN with RPA is Required for Recovery of Replication Forks Stalled at Secondary DNA Structures

    doi: 10.1101/2023.08.08.552428

    Figure Lengend Snippet: A) Ponceau staining of GST-pulldowns with HEK293T nuclear extracts and GST-tagged WRN fragment 403-503 (WRN wt and WRN 6D ) previously phosphorylated by CK2 in the presence or not of ATP. B) Western Blotting analysis of WRN S440/467 phosphorylation and RPA32 subunit from GST-pulldowns. The graph shows the levels of S440/467 phosphorylation and RPA32 bound to GST-tagged WRN fragments. C) Anti-Flag-immunoprecipitation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The graph shows the quantification of the WRN-normalised amount of RPA70 in the anti-Flag immunoprecipitate from the representative experiment. D) Anti-RPA70 immunoprecipitatation from HEK293T cells transfected with Flag-WRN or Flag-WRN6A constructs. The fraction of pS440/467 phosphorylated WRN associated with RPA was analysed using the anti-pS440/467WRN antibody. The graph shows the quantification of the RPA70-normalised amount of WRN in the anti-RPA70 immunoprecipitate from the representative experiment. E) Anti-RPA70 immunoprecipitation from HEK293T cells transfected with Flag-WRN wt construct. Cells were treated with HU 2mM for 2h in the presence or not of the indicated inhibitors. F) WRN/RPA32 interaction was detected in Werner Syndrome (WS) cells nucleofected with Flag-WRN wt or Flag-WRN 6A by in situ Proximity Ligation Assay using anti-FLAG and RPA32 antibodies. The graphs show individual values of PLA spots. Representative images are shown. Bars represent mean ± S.E. (*P<0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001. Where not indicated, values are not significant).

    Article Snippet: Two mL of lysate were incubated overnight at 4°C with 20 μl of Anti-Flag M2 magnetic beads (Sigma) or Anti-RPA32 conjugated Dynabeads (2μg of MABE285 anti-RPA34-20 mouse (Millipore) with 40μl of Dynabeads protein G (Invitrogen).

    Techniques: Staining, Western Blot, Immunoprecipitation, Transfection, Construct, In Situ, Proximity Ligation Assay