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Proteintech rap80

PKMYT1-induced NPM1 S260 phosphorylation promotes efficient DSB repair. a Western blot analysis of the expression of PKMYT1, phosphorylated-NPM1 S260, and phosphorylated-NPM1 T199 in OS cells after being treated with various concentrations of DDP. b – d Western blot analysis of the effect of DDP and IR on NPM1 S260 phosphorylation after PKMYT1 knockdown or inhibition with RP6306. e Western blot analysis of the impact of DDP and IR on NPM1 S260 phosphorylation following the overexpression of NPM1 S260 dephosphorylation mutant plasmid. f After transfection as indicated siRNAs for 48 h, HEK293T cells were collected and subjected to co-immunoprecipitation experiments followed by western blot analysis. g HEK293T cells were transfected with indicated siRNAs for 48 h and treated with or without cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. h HEK293T cells were treated with or without RP6306 (2 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. i HEK293T cells were treated with or without RP6306 (2 μM) and cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. j HEK293T cells were transfected with indicated plasmids for 24 h and treated with or without cisplatin (1 μM) before harvesting the cells for co-immunoprecipitation and Western blot. k – l Immunofluorescence analysis of the effect of NPM1 S260 phosphorylation on IR-induced BRCA1 foci. NPM1 knockout U2OS cells were rescued with either wild-type NPM1, NPM1 S260A mutant and NPM1 S260D mutant, with or without IR (10 Gy) treatment. Representative images are shown in k , and the number of BRCA1 foci per group was calculated in l. Approximately 100 cells were counted per group. Data are expressed as the mean ± SEM from three biological replicates. Statistical analysis was performed using Student’s t test, p value as indicated. Scale bar, 10 μm. m – n Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in m, and the number of BRCA1 foci per group was calculated in n, p value as indicated. Scale bar, 10 μm. o – p NPM1 knockout U2OS cells were rescued with indicated plasmids for 24 h, and immunofluorescence analysis of cells exposed to 10 Gy IR and recovered for 1 h. Representative immunofluorescence images were shown in o , and the number of RAD51 foci per group was shown in p , p value as indicated. Scale bar, 10 μm. q – r Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( q ), and the number of RAD51 foci per group was calculated in r . Scale bar, 10 μm. s , t Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( s ), and the number of <t>RAP80</t> foci per group was calculated in t , p value as indicated. Scale bar, 10 μm. u HR assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with BRCA1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts. P value as indicated. v NHEJ assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with 53BP1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts, p value as indicated

Rap80, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews

rap80 - by Bioz Stars, 2026-06

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1) Product Images from "PKMYT1 kinase ameliorates cisplatin sensitivity in osteosarcoma"

Article Title: PKMYT1 kinase ameliorates cisplatin sensitivity in osteosarcoma

Journal: Signal Transduction and Targeted Therapy

doi: 10.1038/s41392-025-02250-7

Figure Legend Snippet: PKMYT1-induced NPM1 S260 phosphorylation promotes efficient DSB repair. a Western blot analysis of the expression of PKMYT1, phosphorylated-NPM1 S260, and phosphorylated-NPM1 T199 in OS cells after being treated with various concentrations of DDP. b – d Western blot analysis of the effect of DDP and IR on NPM1 S260 phosphorylation after PKMYT1 knockdown or inhibition with RP6306. e Western blot analysis of the impact of DDP and IR on NPM1 S260 phosphorylation following the overexpression of NPM1 S260 dephosphorylation mutant plasmid. f After transfection as indicated siRNAs for 48 h, HEK293T cells were collected and subjected to co-immunoprecipitation experiments followed by western blot analysis. g HEK293T cells were transfected with indicated siRNAs for 48 h and treated with or without cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. h HEK293T cells were treated with or without RP6306 (2 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. i HEK293T cells were treated with or without RP6306 (2 μM) and cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. j HEK293T cells were transfected with indicated plasmids for 24 h and treated with or without cisplatin (1 μM) before harvesting the cells for co-immunoprecipitation and Western blot. k – l Immunofluorescence analysis of the effect of NPM1 S260 phosphorylation on IR-induced BRCA1 foci. NPM1 knockout U2OS cells were rescued with either wild-type NPM1, NPM1 S260A mutant and NPM1 S260D mutant, with or without IR (10 Gy) treatment. Representative images are shown in k , and the number of BRCA1 foci per group was calculated in l. Approximately 100 cells were counted per group. Data are expressed as the mean ± SEM from three biological replicates. Statistical analysis was performed using Student’s t test, p value as indicated. Scale bar, 10 μm. m – n Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in m, and the number of BRCA1 foci per group was calculated in n, p value as indicated. Scale bar, 10 μm. o – p NPM1 knockout U2OS cells were rescued with indicated plasmids for 24 h, and immunofluorescence analysis of cells exposed to 10 Gy IR and recovered for 1 h. Representative immunofluorescence images were shown in o , and the number of RAD51 foci per group was shown in p , p value as indicated. Scale bar, 10 μm. q – r Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( q ), and the number of RAD51 foci per group was calculated in r . Scale bar, 10 μm. s , t Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( s ), and the number of RAP80 foci per group was calculated in t , p value as indicated. Scale bar, 10 μm. u HR assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with BRCA1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts. P value as indicated. v NHEJ assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with 53BP1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts, p value as indicated

Techniques Used: Phospho-proteomics, Western Blot, Expressing, Knockdown, Inhibition, Over Expression, De-Phosphorylation Assay, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Knock-Out, Control, NHEJ Assay

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(a and b), Representative immunofluorescence images of RAP80 foci in HT-29 cells treated with CPT (1 µM) alone or in combination with ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment ( a ). Quantification of the RAP80 foci is shown ( b ). Experiments were performed four times with similar results. Data represent mean ± SEM derived from n ≥ 325 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. c, Representative immunofluorescence images showing increased HA-ABRAXAS foci in HT-29 cells upon ATM inhibition. Cells were treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. γH2AX marks cells with CPT-induced replication damage. Scale bar is shown. d, Quantification of the data from experiments as described in (c). Experiments were repeated three times with similar results. Data are mean ± SEM derived from n ≥ 300 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. (e and f), Representative immunofluorescence images of BRCA1 foci in control (sgROSA) and ABRAXAS KO HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment ( e ). Scatter plot showing ABRAXAS-dependent increased BRCA1 foci in CPT-treated cells following ATM inhibition ( f ). Data represent mean ± SEM derived from n ≥ 145 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test.

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: (a and b), Representative immunofluorescence images of RAP80 foci in HT-29 cells treated with CPT (1 µM) alone or in combination with ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment ( a ). Quantification of the RAP80 foci is shown ( b ). Experiments were performed four times with similar results. Data represent mean ± SEM derived from n ≥ 325 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. c, Representative immunofluorescence images showing increased HA-ABRAXAS foci in HT-29 cells upon ATM inhibition. Cells were treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. γH2AX marks cells with CPT-induced replication damage. Scale bar is shown. d, Quantification of the data from experiments as described in (c). Experiments were repeated three times with similar results. Data are mean ± SEM derived from n ≥ 300 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. (e and f), Representative immunofluorescence images of BRCA1 foci in control (sgROSA) and ABRAXAS KO HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment ( e ). Scatter plot showing ABRAXAS-dependent increased BRCA1 foci in CPT-treated cells following ATM inhibition ( f ). Data represent mean ± SEM derived from n ≥ 145 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test.

Article Snippet: ABRAXAS and RAP80 mutants were generated by site-directed mutagenesis of pOZ-N-FH Abraxas (Addgene, Plasmid #27495) and pOZ-N-FH RAP80 (Addgene, Plasmid #27501) retrovirus expression plasmids, respectively using Q5 Site-Directed Mutagenesis kit (New England Biolabs, E0554S).

Techniques: Immunofluorescence, Derivative Assay, Two Tailed Test, Inhibition, Control

a , Schematic showing structural domains of RAP80. SIM, SUMO interaction motif; UIMs, Ubiquitin interaction motifs; AIR, ABRAXAS interacting region; ZnFs, Zinc finger domains. b , Western blots showing RAP80 protein levels in RAP80 KO HT-29 cells complemented with FLAG-HA tagged WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM RAP80. GAPDH serves as loading control. c, Scatter plot showing quantification of HA-RAP80 foci in WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM expressing HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. Data represent mean ± SEM derived from n ≥ 230 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. d, Scatter plot showing quantification of BRCA1 foci in the genotypes as indicated in (c). Data are mean ± SEM from n ≥ 280 cells examined over two repeats; p values are indicated, unpaired two-tailed t test. e, Quantification of RPA immunofluorescence in HT-29 cells expressing RAP80 WT or the indicated deletion mutants upon treatment with CPT (1 µM) + ATMi (250 nM) for 1 h. Data represent mean ± SEM derived from n ≥ 125 cells examined over two repeats; p values are indicated, unpaired two-tailed test. f, Line graph showing relative colony survival of control (sgROSA) and RAP80 KO HT-29 cells harboring empty vector (EV) or complemented with WT or RAP80 deletion mutants. Cells were treated with 100 nM ATMi and the indicated doses of CPT. p values were determined for differences in colony survival for RAP80 KO (EV), ΔSIM or ΔSIM + ΔUIM harboring cells over WT at CPT concentrations of 0.5 and 1 nM; unpaired two-tailed t test. Data represents mean ± SD from n =3 biological experiments. g, Scatter plot showing quantification of RAP80-EdU PLA in control (sgROSA), RNF8 or RNF168 KO HT-29 cells treated with 50 nM CPT +/- ATMi (250 nM). Cells were optionally pretreated with ATMi for 10 minutes followed by CPT treatment for 40 minutes. EdU was added during the last 20 min of CPT treatment. ATMi was present throughout the experiment. Data represent mean ± SEM derived from n ≥ 279 nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test. h, Quantification of BRCA1-EdU PLA as described in (e). Data are mean ± SEM from n ≥ 239 nuclei pooled from two independent experiments; p values are indicated, unpaired two-tailed t test.

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a , Schematic showing structural domains of RAP80. SIM, SUMO interaction motif; UIMs, Ubiquitin interaction motifs; AIR, ABRAXAS interacting region; ZnFs, Zinc finger domains. b , Western blots showing RAP80 protein levels in RAP80 KO HT-29 cells complemented with FLAG-HA tagged WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM RAP80. GAPDH serves as loading control. c, Scatter plot showing quantification of HA-RAP80 foci in WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM expressing HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. Data represent mean ± SEM derived from n ≥ 230 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. d, Scatter plot showing quantification of BRCA1 foci in the genotypes as indicated in (c). Data are mean ± SEM from n ≥ 280 cells examined over two repeats; p values are indicated, unpaired two-tailed t test. e, Quantification of RPA immunofluorescence in HT-29 cells expressing RAP80 WT or the indicated deletion mutants upon treatment with CPT (1 µM) + ATMi (250 nM) for 1 h. Data represent mean ± SEM derived from n ≥ 125 cells examined over two repeats; p values are indicated, unpaired two-tailed test. f, Line graph showing relative colony survival of control (sgROSA) and RAP80 KO HT-29 cells harboring empty vector (EV) or complemented with WT or RAP80 deletion mutants. Cells were treated with 100 nM ATMi and the indicated doses of CPT. p values were determined for differences in colony survival for RAP80 KO (EV), ΔSIM or ΔSIM + ΔUIM harboring cells over WT at CPT concentrations of 0.5 and 1 nM; unpaired two-tailed t test. Data represents mean ± SD from n =3 biological experiments. g, Scatter plot showing quantification of RAP80-EdU PLA in control (sgROSA), RNF8 or RNF168 KO HT-29 cells treated with 50 nM CPT +/- ATMi (250 nM). Cells were optionally pretreated with ATMi for 10 minutes followed by CPT treatment for 40 minutes. EdU was added during the last 20 min of CPT treatment. ATMi was present throughout the experiment. Data represent mean ± SEM derived from n ≥ 279 nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test. h, Quantification of BRCA1-EdU PLA as described in (e). Data are mean ± SEM from n ≥ 239 nuclei pooled from two independent experiments; p values are indicated, unpaired two-tailed t test.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Expressing, Derivative Assay, Two Tailed Test, Immunofluorescence, Plasmid Preparation

a, Representative immunofluorescence images of HA-RAP80 foci in WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM expressing HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. b, Representative BRCA1 foci in WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM expressing HT-29 cells treated as described in (a). (c, d) Inhibition of SUMOylation by the SUMO E1 inhibitor ML-792 reduces BRCA1-A damage localization in ATMi-treated cells. Representative images of RAP80-EdU PLA in HT-29 cells treated with 50 nM CPT+/- 250 nM ATMi. Cells were optionally treated with ML-792 (1 µM) for 16 h prior to CPT treatment (c). Quantification of RAP80-SIRF PLA foci is shown (d). Data represent mean ± SEM derived from n ≥ 545 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. e, Left panel: PLA-SIRF experiment showing increased SUMO2/3 abundance at CPT-damaged fork upon ATM inhibition. HT-29 cells were optionally pretreated with ATMi (250 nM) for 10 minutes followed by CPT (1 µM) treatment for 40 minutes. EdU was added during the last 20 min of CPT treatment. ATMi was present throughout the experiment. Right panel: Scatter plot showing quantification of SUMO2/3-EdU SIRF foci normalized to average EdU-EdU signal in each experimental condition. Experiments were performed three times with similar results. Data represent mean ± SEM derived from n ≥ 500 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. f, Left panel: PLA-SIRF experiment showing FK2-Ub accumulation at CPT-damaged fork upon ATM inhibition. HT-29 cells were treated as described in (e). Right panel: Quantification of the data is shown. Experiments were performed four times with similar results. Data represent mean ± SEM derived from n ≥ 506 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test.

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a, Representative immunofluorescence images of HA-RAP80 foci in WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM expressing HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. b, Representative BRCA1 foci in WT, ΔUIM, ΔSIM or ΔSIM + ΔUIM expressing HT-29 cells treated as described in (a). (c, d) Inhibition of SUMOylation by the SUMO E1 inhibitor ML-792 reduces BRCA1-A damage localization in ATMi-treated cells. Representative images of RAP80-EdU PLA in HT-29 cells treated with 50 nM CPT+/- 250 nM ATMi. Cells were optionally treated with ML-792 (1 µM) for 16 h prior to CPT treatment (c). Quantification of RAP80-SIRF PLA foci is shown (d). Data represent mean ± SEM derived from n ≥ 545 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. e, Left panel: PLA-SIRF experiment showing increased SUMO2/3 abundance at CPT-damaged fork upon ATM inhibition. HT-29 cells were optionally pretreated with ATMi (250 nM) for 10 minutes followed by CPT (1 µM) treatment for 40 minutes. EdU was added during the last 20 min of CPT treatment. ATMi was present throughout the experiment. Right panel: Scatter plot showing quantification of SUMO2/3-EdU SIRF foci normalized to average EdU-EdU signal in each experimental condition. Experiments were performed three times with similar results. Data represent mean ± SEM derived from n ≥ 500 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. f, Left panel: PLA-SIRF experiment showing FK2-Ub accumulation at CPT-damaged fork upon ATM inhibition. HT-29 cells were treated as described in (e). Right panel: Quantification of the data is shown. Experiments were performed four times with similar results. Data represent mean ± SEM derived from n ≥ 506 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test.

Techniques: Immunofluorescence, Expressing, Inhibition, Derivative Assay, Two Tailed Test

a, Representative RPA immunofluorescence in control (sgROSA) and RAP80 KO HT-29 cells complemented with WT or RAP80 deletion mutants. Cells were pretreated with ATMi (250 nM) for 10 min followed by CPT (1 µM) treatment for 1 h. b, Representative colony images of control (sgROSA) or RAP80 KO HT-29 cells harboring empty vector (EV) or complemented with WT or RAP80 deletion mutants, Cells were treated with 100 nM ATMi in combination with the indicated doses of CPT.

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a, Representative RPA immunofluorescence in control (sgROSA) and RAP80 KO HT-29 cells complemented with WT or RAP80 deletion mutants. Cells were pretreated with ATMi (250 nM) for 10 min followed by CPT (1 µM) treatment for 1 h. b, Representative colony images of control (sgROSA) or RAP80 KO HT-29 cells harboring empty vector (EV) or complemented with WT or RAP80 deletion mutants, Cells were treated with 100 nM ATMi in combination with the indicated doses of CPT.

Techniques: Immunofluorescence, Control, Plasmid Preparation

(a,b) Scatter plot showing CldU (a) and IdU (b) tract length determined from the fork restart experiments as described in . p -values were determined from n ≥ 135 DNA fibers using two-tailed Mann–Whitney test. Red bar indicates median tract length. c, Bar graph showing quantification of chromosomal aberrations in HT-29 cells expressing either WT or SIM+UIM deleted RAP80. Data represent mean ± SEM of n=3 repeats. At least 60 metaphase spreads were analyzed for each condition across three repeats; p values are indicated; unpaired two-tailed t test.

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: (a,b) Scatter plot showing CldU (a) and IdU (b) tract length determined from the fork restart experiments as described in . p -values were determined from n ≥ 135 DNA fibers using two-tailed Mann–Whitney test. Red bar indicates median tract length. c, Bar graph showing quantification of chromosomal aberrations in HT-29 cells expressing either WT or SIM+UIM deleted RAP80. Data represent mean ± SEM of n=3 repeats. At least 60 metaphase spreads were analyzed for each condition across three repeats; p values are indicated; unpaired two-tailed t test.

Techniques: Two Tailed Test, MANN-WHITNEY, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: PKMYT1 kinase ameliorates cisplatin sensitivity in osteosarcoma

doi: 10.1038/s41392-025-02250-7

Figure Lengend Snippet: PKMYT1-induced NPM1 S260 phosphorylation promotes efficient DSB repair. a Western blot analysis of the expression of PKMYT1, phosphorylated-NPM1 S260, and phosphorylated-NPM1 T199 in OS cells after being treated with various concentrations of DDP. b – d Western blot analysis of the effect of DDP and IR on NPM1 S260 phosphorylation after PKMYT1 knockdown or inhibition with RP6306. e Western blot analysis of the impact of DDP and IR on NPM1 S260 phosphorylation following the overexpression of NPM1 S260 dephosphorylation mutant plasmid. f After transfection as indicated siRNAs for 48 h, HEK293T cells were collected and subjected to co-immunoprecipitation experiments followed by western blot analysis. g HEK293T cells were transfected with indicated siRNAs for 48 h and treated with or without cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. h HEK293T cells were treated with or without RP6306 (2 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. i HEK293T cells were treated with or without RP6306 (2 μM) and cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. j HEK293T cells were transfected with indicated plasmids for 24 h and treated with or without cisplatin (1 μM) before harvesting the cells for co-immunoprecipitation and Western blot. k – l Immunofluorescence analysis of the effect of NPM1 S260 phosphorylation on IR-induced BRCA1 foci. NPM1 knockout U2OS cells were rescued with either wild-type NPM1, NPM1 S260A mutant and NPM1 S260D mutant, with or without IR (10 Gy) treatment. Representative images are shown in k , and the number of BRCA1 foci per group was calculated in l. Approximately 100 cells were counted per group. Data are expressed as the mean ± SEM from three biological replicates. Statistical analysis was performed using Student’s t test, p value as indicated. Scale bar, 10 μm. m – n Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in m, and the number of BRCA1 foci per group was calculated in n, p value as indicated. Scale bar, 10 μm. o – p NPM1 knockout U2OS cells were rescued with indicated plasmids for 24 h, and immunofluorescence analysis of cells exposed to 10 Gy IR and recovered for 1 h. Representative immunofluorescence images were shown in o , and the number of RAD51 foci per group was shown in p , p value as indicated. Scale bar, 10 μm. q – r Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( q ), and the number of RAD51 foci per group was calculated in r . Scale bar, 10 μm. s , t Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( s ), and the number of RAP80 foci per group was calculated in t , p value as indicated. Scale bar, 10 μm. u HR assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with BRCA1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts. P value as indicated. v NHEJ assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with 53BP1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts, p value as indicated

Article Snippet: The antibodies used in this study are as follows: GAPDH (#60004-1-Ig, Proteintech, 1:10,000 dilution), PKMYT1 (#4282S, Cell Signaling Technology, 1:1,000 dilution), NPM1 (#60096-1-Ig, Proteintech, 1:5,000 dilution), p-NPM1 T199 (#AF3111, Affnity, 1:1000 dilution), p-NPM1 S260 (#TP50488, HUABIO, 1:3000 dilution), p-Histone H2A.X S139 (# P40705 , ProMab Biotechnologies Inc., China, 1:1000 dilution), RAP80 (#13642-1-AP, Proteintech, 1:1000 dilution), BRCA1 (#22362-1-AP, Proteintech, 1:1000 dilution), RAD51(#14961-1-AP, Proteintech, China, 1:2000 dilution), anti-HA (#AB0004, Abways, China, 1:2000 dilution), β-actin (A00730, Genscript Biotech, 1:1000 dilution), Phosphoserine/threonine/tyrosine (#11995 R, Yajikit, 1:100 dilution), HRP conjugated goat-anti-mouse antibody (#SA00001-1, Proteintech, 1:10,000 dilution) and HRP conjugated goat-anti-rabbit antibody (#511203, zen-bioscience, 1:5000 dilution).

Techniques: Phospho-proteomics, Western Blot, Expressing, Knockdown, Inhibition, Over Expression, De-Phosphorylation Assay, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Knock-Out, Control, NHEJ Assay

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