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DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and <t>DHX9/RPA32(</t> B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.
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DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and <t>DHX9/RPA32(</t> B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.
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DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and <t>DHX9/RPA32(</t> B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.
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DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and <t>DHX9/RPA32(</t> B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.
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DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and <t>DHX9/RPA32(</t> B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.
26418 Candida Kefyr, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and DHX9/RPA32( B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.

Journal: Nucleic Acids Research

Article Title: ATR phosphorylates DHX9 at serine 321 to suppress R-loop accumulation upon genotoxic stress

doi: 10.1093/nar/gkad973

Figure Lengend Snippet: DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation induced by DNA damage. (A, B) The association between DHX9/γH2AX ( A ) and DHX9/RPA32( B ) in HeLa cells. Cells transfected with control or DHX9 siRNA were treated with DMSO or CPT (1 μM, 1 h), analyzed by PLA with indicated antibodies. Representative images were shown, and the numbers of PLA foci were obtained from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) A primary structure of DHX9 and sequence alignment of conserved S321 and S688 across human, bovine, mouse and Xenopus. ( D ) HeLa cells transiently expressed SFB-DHX9 variants (WT, S321A and AA) were subjected to treatment with or without HU (2 mM) for 1 h. SFB-DHX9 (WT, S321A and AA) were immunoprecipitated by an anti-FLAG antibody under a denaturing condition. Levels of DHX9 phosphorylation at S/TQ sites and protein expression were determined by Western blot with indicated antibodies. (E, F) Time course of DHX9 phosphorylation at S321 was determined by a phospho-specific antibody to this site. HeLa cells irradiated by UV (20 J/m 2 ) ( E ) or treated with CPT (10 μM) ( F ) were collected at a series of time points, and phosphorylation of DHX9 pS321 and Chk1 pS317 after DNA damage was accessed by Western blot with indicated antibodies. ( G ) HeLa cells treated with CPT (1 μM) or left untreated were analyzed by Western blot with indicated antibodies. ( H ) Validation of the specificity of anti-DHX9 pS321 antibody. HeLa cells devoid of DHX9 by siRNA were transfected with siDHX9-1 insensitive SFB-DHX9 WT or SFB-DHX9 S321A . Cells were treated with or without CPT (1 μM, 1 h), and the phosphorylation of S321 was determined by Western blot with indicated antibodies. ( I ) Phosphorylation at S688 was determined by a phospho-specific antibody to this site. HeLa cells transfected with SFB-DHX9 WT , SFB-DHX9 S688A or the corresponding empty vector were analyzed by Western blot with indicated antibodies.

Article Snippet: The antibodies were used where indicated. pChk1 S317(#12302), pChk1 S345 (#2348), pChk2(#2661), Chk2 (#3440), [pS/pT] QG (#6966) and γH2AX (#9718) were obtained from Cell Signaling Technology; ATR (A300-138A), ATM (A300-299A), Rad17 pS645 (A300-153A), RPA32 (A300-244A), pRPA32 S33 (A300-246A) and pRPA32 S4/8 (A300-245A) antibodies were from Bethyl Lab; BRCA1 (sc-6954), DHX9 (sc-137232), FLAG (sc-166355), Chk1 (sc-8408), PARP1 (sc-8007), PRP19 (sc-514338), PCNA (sc-56) and GAPDH (sc-47724) antibodies from Santa Cruz; DHX9 (ab26271) were purchased from abcam; FLAG (F7425), γH2AX (05–636), α-Tubulin (T6074) and S9.6 (MABE1095) were purchased from Merck; RPA32 (MA1-26418), goat anti-mouse-HRP (G21040), goat anti-rabbit-HRP (G21234), goat anti-mouse Alexa Fluor-594 (A-11005) and goat anti-rabbit Alexa Fluor-488 (A-11034) were from ThermoFisher; pATR (GTX128145), pATM (GTX132146), RPA70 (GTX108749) were obtained from GeneTex; HRP conjugated anti-mouse (115-035-174) and anti-rabbit (211-032-171), light chain specific antibodies were obtained from Jackson ImmunoResearch; affinity-purified DHX9 pS321 and pS688 were customized by GeneTex.

Techniques: Transfection, MANN-WHITNEY, Sequencing, Immunoprecipitation, Expressing, Western Blot, Irradiation, Plasmid Preparation

Phosphorylation of DHX9 promotes its interaction with RPA. (A, B) The association between DHX9 pS321/γH2AX ( A ) and DHX9 pS321/RPA32 ( B ) in HeLa cells treated with CPT (1 μM, 1 h) or left untreated were analyzed by PLA with indicated antibodies. Representative images were shown, and numbers of PLA foci per cell were counted from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) Interaction between RPA and DHX9 was enhanced after DNA damage. HeLa cells transfected with SFB-DHX9 or the corresponding empty vector were treated with DMSO or CPT (1 μM, 1 h). The nuclei were extracted, followed by the IP of nuclear RPA32 using the anti-RPA32 antibody. Co-purification of SFB-DHX9 and RPA70 by RPA32 was determined by Western blot with the designated antibodies. The levels of SFB-DHX9 co-purified were quantified and data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed by the Student's t -test (two-sided). * P < 0.05. ( D ) The impact of ATR-mediated DHX9 phosphorylation on the DHX9/RPA32 association was analyzed by PLA. HeLa cells either treated with 1 μM of VE822 30 min before or 30 min after CPT (1 μM) treatment were analyzed by PLA with indicated antibodies. ( E ) Substitution of phosphorylated serine with alanine for single or double mutations (S321A, S688A, AA) impeded the DHX9/RPA32 interaction after CPT treatment. HeLa cells transfected with the corresponding empty vector (Vect) or SFB-DHX9 variants (WT, S321A, S688A and AA) were treated with CPT (1 μM) for 1 h, and the PLA signal was determined. Representative images were shown, and numbers of PLA foci per cell were counted from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided).*** P < 0.001.

Journal: Nucleic Acids Research

Article Title: ATR phosphorylates DHX9 at serine 321 to suppress R-loop accumulation upon genotoxic stress

doi: 10.1093/nar/gkad973

Figure Lengend Snippet: Phosphorylation of DHX9 promotes its interaction with RPA. (A, B) The association between DHX9 pS321/γH2AX ( A ) and DHX9 pS321/RPA32 ( B ) in HeLa cells treated with CPT (1 μM, 1 h) or left untreated were analyzed by PLA with indicated antibodies. Representative images were shown, and numbers of PLA foci per cell were counted from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided). *** P < 0.001. ( C ) Interaction between RPA and DHX9 was enhanced after DNA damage. HeLa cells transfected with SFB-DHX9 or the corresponding empty vector were treated with DMSO or CPT (1 μM, 1 h). The nuclei were extracted, followed by the IP of nuclear RPA32 using the anti-RPA32 antibody. Co-purification of SFB-DHX9 and RPA70 by RPA32 was determined by Western blot with the designated antibodies. The levels of SFB-DHX9 co-purified were quantified and data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed by the Student's t -test (two-sided). * P < 0.05. ( D ) The impact of ATR-mediated DHX9 phosphorylation on the DHX9/RPA32 association was analyzed by PLA. HeLa cells either treated with 1 μM of VE822 30 min before or 30 min after CPT (1 μM) treatment were analyzed by PLA with indicated antibodies. ( E ) Substitution of phosphorylated serine with alanine for single or double mutations (S321A, S688A, AA) impeded the DHX9/RPA32 interaction after CPT treatment. HeLa cells transfected with the corresponding empty vector (Vect) or SFB-DHX9 variants (WT, S321A, S688A and AA) were treated with CPT (1 μM) for 1 h, and the PLA signal was determined. Representative images were shown, and numbers of PLA foci per cell were counted from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by the Mann–Whitney test (two-sided).*** P < 0.001.

Article Snippet: The antibodies were used where indicated. pChk1 S317(#12302), pChk1 S345 (#2348), pChk2(#2661), Chk2 (#3440), [pS/pT] QG (#6966) and γH2AX (#9718) were obtained from Cell Signaling Technology; ATR (A300-138A), ATM (A300-299A), Rad17 pS645 (A300-153A), RPA32 (A300-244A), pRPA32 S33 (A300-246A) and pRPA32 S4/8 (A300-245A) antibodies were from Bethyl Lab; BRCA1 (sc-6954), DHX9 (sc-137232), FLAG (sc-166355), Chk1 (sc-8408), PARP1 (sc-8007), PRP19 (sc-514338), PCNA (sc-56) and GAPDH (sc-47724) antibodies from Santa Cruz; DHX9 (ab26271) were purchased from abcam; FLAG (F7425), γH2AX (05–636), α-Tubulin (T6074) and S9.6 (MABE1095) were purchased from Merck; RPA32 (MA1-26418), goat anti-mouse-HRP (G21040), goat anti-rabbit-HRP (G21234), goat anti-mouse Alexa Fluor-594 (A-11005) and goat anti-rabbit Alexa Fluor-488 (A-11034) were from ThermoFisher; pATR (GTX128145), pATM (GTX132146), RPA70 (GTX108749) were obtained from GeneTex; HRP conjugated anti-mouse (115-035-174) and anti-rabbit (211-032-171), light chain specific antibodies were obtained from Jackson ImmunoResearch; affinity-purified DHX9 pS321 and pS688 were customized by GeneTex.

Techniques: MANN-WHITNEY, Transfection, Plasmid Preparation, Copurification, Western Blot, Purification

DHX9 phosphorylation at S321 enhances its association with RPA. ( A ) Depletion of RPA impeded the association between DHX9 and γH2AX. HeLa cells transfected with control or RPA32 siRNA were then treated with DMSO or CPT (1 μM) for 1 h. The association between DHX9 and γH2AX was assessed by PLA using the indicated antibodies. Representative images were shown, and numbers of PLA foci were quantified from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by Student's t -test (two-sided). *** P < 0.001. (B, C) Ablation of S321 phosphorylation attenuated the interaction between DHX9 and RPA. HeLa cells transfected with SFB-DHX9 WT and SFB-DHX9 S321A were treated with CPT (1 μM) for 1 h. Endogenous RPA32 and SFB-DHX9 were immunoprecipitated from nuclear extracts with anti-RPA32 and anti-FLAG antibodies, respectively. Proteins coprecipitated by RPA32 ( B ) and SFB-DHX9 ( C ) were assessed by Western blot with indicated antibodies. (D, E) The interaction between DHX9 and RPA was not dependent on DNA or RNA. As in (B) and (C), nuclear extracts were pre-incubated with Universal Nuclease before IP of RPA32 ( D ) and SFB-DHX9 ( E ). Proteins coprecipitated by RPA32 (D) and SFB-DHX9 (E) were assessed by Western blot with indicated antibodies. ( F ) A workflow of SFB-DHX9 purification and in vitro SFB-DHX9/RPA binding analysis. ( G, H ) Purified SFB-DHX9 directly interacted with recombinant RPA. HeLa cells transiently expressing SFB-DHX9 were treated with vehicle, CPT (1 μM, 1 h) or HU (4 mM, 2 h) prior to the purification of SFB-DHX9 using anti-FLAG beads. The purified SFB-DHX9 proteins were incubated with 100 ng of the RPA complex for in vitro binding assay. The interaction between SFB-DHX9 and RPA was determined by Western blot with indicated antibodies.

Journal: Nucleic Acids Research

Article Title: ATR phosphorylates DHX9 at serine 321 to suppress R-loop accumulation upon genotoxic stress

doi: 10.1093/nar/gkad973

Figure Lengend Snippet: DHX9 phosphorylation at S321 enhances its association with RPA. ( A ) Depletion of RPA impeded the association between DHX9 and γH2AX. HeLa cells transfected with control or RPA32 siRNA were then treated with DMSO or CPT (1 μM) for 1 h. The association between DHX9 and γH2AX was assessed by PLA using the indicated antibodies. Representative images were shown, and numbers of PLA foci were quantified from at least 150 nuclei in each condition. Data are presented in scatter plots with the black line indicating the median, and statistical significance was assessed by Student's t -test (two-sided). *** P < 0.001. (B, C) Ablation of S321 phosphorylation attenuated the interaction between DHX9 and RPA. HeLa cells transfected with SFB-DHX9 WT and SFB-DHX9 S321A were treated with CPT (1 μM) for 1 h. Endogenous RPA32 and SFB-DHX9 were immunoprecipitated from nuclear extracts with anti-RPA32 and anti-FLAG antibodies, respectively. Proteins coprecipitated by RPA32 ( B ) and SFB-DHX9 ( C ) were assessed by Western blot with indicated antibodies. (D, E) The interaction between DHX9 and RPA was not dependent on DNA or RNA. As in (B) and (C), nuclear extracts were pre-incubated with Universal Nuclease before IP of RPA32 ( D ) and SFB-DHX9 ( E ). Proteins coprecipitated by RPA32 (D) and SFB-DHX9 (E) were assessed by Western blot with indicated antibodies. ( F ) A workflow of SFB-DHX9 purification and in vitro SFB-DHX9/RPA binding analysis. ( G, H ) Purified SFB-DHX9 directly interacted with recombinant RPA. HeLa cells transiently expressing SFB-DHX9 were treated with vehicle, CPT (1 μM, 1 h) or HU (4 mM, 2 h) prior to the purification of SFB-DHX9 using anti-FLAG beads. The purified SFB-DHX9 proteins were incubated with 100 ng of the RPA complex for in vitro binding assay. The interaction between SFB-DHX9 and RPA was determined by Western blot with indicated antibodies.

Article Snippet: The antibodies were used where indicated. pChk1 S317(#12302), pChk1 S345 (#2348), pChk2(#2661), Chk2 (#3440), [pS/pT] QG (#6966) and γH2AX (#9718) were obtained from Cell Signaling Technology; ATR (A300-138A), ATM (A300-299A), Rad17 pS645 (A300-153A), RPA32 (A300-244A), pRPA32 S33 (A300-246A) and pRPA32 S4/8 (A300-245A) antibodies were from Bethyl Lab; BRCA1 (sc-6954), DHX9 (sc-137232), FLAG (sc-166355), Chk1 (sc-8408), PARP1 (sc-8007), PRP19 (sc-514338), PCNA (sc-56) and GAPDH (sc-47724) antibodies from Santa Cruz; DHX9 (ab26271) were purchased from abcam; FLAG (F7425), γH2AX (05–636), α-Tubulin (T6074) and S9.6 (MABE1095) were purchased from Merck; RPA32 (MA1-26418), goat anti-mouse-HRP (G21040), goat anti-rabbit-HRP (G21234), goat anti-mouse Alexa Fluor-594 (A-11005) and goat anti-rabbit Alexa Fluor-488 (A-11034) were from ThermoFisher; pATR (GTX128145), pATM (GTX132146), RPA70 (GTX108749) were obtained from GeneTex; HRP conjugated anti-mouse (115-035-174) and anti-rabbit (211-032-171), light chain specific antibodies were obtained from Jackson ImmunoResearch; affinity-purified DHX9 pS321 and pS688 were customized by GeneTex.

Techniques: Transfection, Immunoprecipitation, Western Blot, Incubation, Purification, In Vitro, Binding Assay, Recombinant, Expressing