Journal: Cell Reports

Article Title: Phospho-Ser 784 -VCP Is Required for DNA Damage Response and Is Associated with Poor Prognosis of Chemotherapy-Treated Breast Cancer

doi: 10.1016/j.celrep.2020.107745

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-pSer 345 -Chk1 , Cell Signaling Technology , Cat#2348; RRID: AB_331212.

Techniques: Recombinant, Single Cell Gel Electrophoresis, Plasmid Preparation, Software, Lysis

Journal: Cell Reports

Article Title: Phospho-Ser 784 -VCP Is Required for DNA Damage Response and Is Associated with Poor Prognosis of Chemotherapy-Treated Breast Cancer

doi: 10.1016/j.celrep.2020.107745

Figure Lengend Snippet:

Article Snippet: Commercial antibodies used for western blot include GFP (CST, #2956), VCP (Santa Cruz, sc-57492 and sc-136273), actin (Santa Cruz, sc-47778), GAPDH (Santa Cruz, sc-365062), histone H3 (CST, #4499), tubulin (Sigma, T9026), PELP1 (Bethyl, #A300-180A), γH2AX (CST, #9718), Ku70 (Santa Cruz, sc-17789), ATM (Santa Cruz, sc-377293), pSer 1981 -ATM (CST, #5883; Rockland, #200-301-400), pSer 428 -ATR (CST, #2853), ATR (CST, #13934), Chk2 (CST, #3440), pThr 68 -Chk2 (CST, #2197), pSer 345 -Chk1 (CST, #2348), NBS1 (CST, #14956), pSer 343 -NBS1 (CST, #3011), K48-ubiquitin (CST, #4289; CST, #8081), FLAG tag (BioLegend, #637301), NPL4 (Bethyl, #A304-102A), UFD1 (Bethyl, #A301-875A), CHOP (CST, #2895), BiP (CST, #3177), phospho-ATM/ATR substrate motif (CST, #6966), Pfn1 (CST, #3246), HIF1α (CST, #79233).

Techniques: Recombinant, Single Cell Gel Electrophoresis, Plasmid Preparation, Software, Lysis

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: The specificity of anti–phospho-Ser-280 on Chk1 (αpS280) or anti-Chk1 (αChk1). (A) After 48 h of serum starvation, RPE1 cells were incubated with the fresh growing medium for 0 or 10 min as described in Materials and Methods . Specificity of each antibody was analyzed by immunoblotting. Amounts of loading cell lysates were determined by staining of SDS–PAGE gel with Coomassie brilliant blue (CBB; left). In the competition assay, αpS280 was preincubated with 50 ng/ml nonphosphopeptide S280 or each phosphopeptide (pS), and then the serum-stimulated cell lysate was immunoblotted (right). (B) Cells were stained with αpS280 (green), αChk1 (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue; left). The N/C ratio of αChk1 intensity is shown. Data represent mean ± SEM for at least 20 cells in each cell group, **p < 0.01 using Student's t test (right). (C) RPE1 cells were transfected with the mixture of the indicated siRNA and Lipofectamine RNAiMAX reagent according to the reverse transfection procedures (Invitrogen). At 16 h after the transfection, the cells were cultured in the serum-free medium for 48 h and then incubated in the growing medium for 10 min. Similar diminishment of the antibody signals in the immunocytochemistry was observed in cells transfected with siChk1 Sq. 2 (unpublished data). Scale bar, 10 μm (B, C).

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Incubation, Western Blot, Staining, SDS Page, Competitive Binding Assay, Transfection, Cell Culture, Immunocytochemistry

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: Chk1 is phosphorylated specifically at Ser-280 in response to serum stimulation. (A, B) Endogenous Chk1 was immunoprecipitated from cells stimulated by 10% serum for 0 or 10 min, HU-treated or mitotic (M) cells. Each immunoprecipitate was subjected to the SDS–PAGE with (B) or without (A) Mn 2+ -Phos-tag, followed by immunoblotting with the indicated antibody. (C) Establishment of each Tet-On RPE1 cell line. Cells were treated with (+) or without (−) 2 ng/ml doxycycline (Dox) for 48 h. SA or SE indicates Myc-tagged Chk1 mutated at Ser-280 to Ala or Glu, respectively. (D) Tet-On RPE1 cell line was cultured in the serum-free medium containing 5 ng/ml Dox for 48 h. After serum starvation, cells were incubated in the growing medium for 0 or 10 min. After treatment, cells were subjected to αMyc immunoprecipitation. The immunoprecipitate (αMyc IP) or a fraction of each cell extract (Input) was subjected to the SDS–PAGE with (+) or without (−) Mn 2+ -Phos-tag, followed by immunoblotting, respectively. (E–I) Each Tet-On cell line was transfected with control or Chk1 3′ UTR siRNA according to the forward transfection procedures (Invitrogen). At 4 h after transfection, the medium was replaced with the fresh growing medium containing Dox. At 24 h after transfection, cells were analyzed by immunoblotting (E, G) or immunocytochemistry (F, H, I). In E, we used Tet-On RPE1 cell line expressing EGFP as a negative control. In G, each Tet-On cell line was also incubated with or without Dox for 24 h in order to evaluate inducible expression of each Myc-Chk1. The N/C ratio of αMyc intensity is shown. Data represent mean ± SEM for at least 20 cells in each cell group, **p < 0.01 vs. WT-replacing cells (I). Similar results were obtained using another Chk1 3′UTR sequence (unpublished data). Scale bar, 10 μm (F, H).

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Cell Culture, Incubation, Transfection, Immunocytochemistry, Expressing, Negative Control, Sequencing

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: Chk1–Ser-280 is phosphorylated by p90 RSK in response to serum stimulation. (A) After 48 h of serum starvation, RPE1 cells were incubated with the growing medium for the indicated time. (B) Schema shows a kinase that each chemical agent inhibits in MAP kinase and PI3-K–Akt/PKB pathways. (C) RPE1 cells were treated with 10 μM U0126, 10 μM LY294002, 10 μM BI-D1870, 1 μM MK-2206, or equal volume of DMSO (control) as described in Materials and Methods . At 5 min after serum addition, cells were analyzed by immunoblotting. (D) HeLa cells were transfected with p90 RSK1/2/3 or Akt1/2 (5 nM of Dharmacon ON-TARGET plus SMARTpool per each protein) according to the reverse transfection procedures (Invitrogen). As a negative control, we used 15 nM of Dharmacon ON-TARGET plus siCONTROL. At 16 h after transfection, cells were cultured in DMEM medium containing 0.5% FBS for 48 h and then incubated in the medium containing 10% FBS for 5 min.

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Incubation, Western Blot, Transfection, Negative Control, Cell Culture

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: Chk1 is translocated from cytoplasm to nucleus in a p90 RSK–dependent manner. RPE1 (A), U2OS, or HeLa (B, C) cells were treated with each chemical agent as described in Materials and Methods . At 5 (A) or 10 (B) min after serum addition, cells were stained with αpS280 (green), αChk1 (red), and DAPI (blue). In C, treated cells were also analyzed by immunoblotting. The N/C ratio of αChk1 intensity is also shown. Data represent mean ± SEM for at least 20 cells in each cell group, **p < 0.01 vs. cells treated with DMSO (A, B). Scale bar, 10 μm (A, B).

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Staining, Western Blot

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: P90 RSK phosphorylates Ser-280 on Chk1. (A–C) Tet-On RPE1 cell line was cultured in the serum-free medium for 48 h. After treatment, cells were cultured in serum-free medium with (+) or without (−) Dox for 6 h. Cells were analyzed by immunoblotting (A) or immunocytochemistry (B, C). CA or KD indicates a constitutively active or kinase-dead mutant, respectively. The N/C ratio of αChk1 intensity is shown. Data represent mean ± SEM for at least 20 cells in each cell group, **p < 0.01 vs. RSK2 KD-expressing cells (C; also see Supplemental Figure S1). Bar, 10 μm (B, C). (D) Purified Chk1 mutant KD (S) or KD/S280A (A) was incubated with or without p90 RSK1 or Akt1 for 20 min as described in Materials and Methods . The reaction mixture was analyzed by SDS–PAGE or immunoblotting with αpS280 (Chk1–pSer-280) or αChk1 (Chk1). After SDS–PAGE, bands of Chk1 or radioactive Chk1 were visualized by staining with CBB or autoradiography ( 32 P), respectively. (E) Time course of Chk1 KD phosphorylation by p90 RSK1.

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Cell Culture, Western Blot, Immunocytochemistry, Mutagenesis, Expressing, Purification, Incubation, SDS Page, Staining, Autoradiography

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: UV irradiation elevates Chk1–Ser-280 phosphorylation level in a p90 RSK–dependent manner. (A–C) RPE1 cells were treated with each genotoxic stimulus as follows. For UV irradiation (A–C), the culture medium was removed, and cells were irradiated in uncovered tissue culture dishes with 254-nm UV light at a dose of 20 J/m 2 (FUNA-UV-LINKER; Funakoshi, Tokyo, Japan). After the growing medium was added back, cells were cultured for 30 min. For IR (A), cells were treated with 20 Gy of x-rays and then cultured for 30 min. For HU treatment (A), cells were treated as described in Materials and Methods . These treated cells were subjected to immunoblotting (A), immunocytochemistry (B), or immunoprecipitation (C). Bar, 10 μm (B). (D, E) RPE1 cells were pretreated with 10 μM U0126, 20 μM SB203580 (D), 10 μM BI-D1870, 1 μM MK-2206 (E), or an equal volume of DMSO (Control) for 30 min. After UV irradiation, cells were incubated in the growing medium containing the same chemical agent for 20 min. The phosphorylation of MK2 (D) or Akt/PKB (E) was also monitored as a control for SB203580 or MK-2206, respectively. Amounts of Chk1 phosphorylated at Ser-280, Ser-296, and Ser-345 were quantified using densitometry, normalized to the content of Chk1, and presented as fold of treatment with DMSO. Data represent mean ± SEM of three independent experiments, *p < 0.05 and **p < 0.01 vs. the treatment with DMSO (E).

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Irradiation, Cell Culture, Western Blot, Immunocytochemistry, Immunoprecipitation, Incubation

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: P90 RSK facilitates Chk1 phosphorylation not only at Ser-280 but also at Ser-296 by Chk1 and at Ser-345 by ATR after UV irradiation. (A–C) Serum starvation, agent treatment, and serum stimulation were performed as described in Materials and Methods . At 10 min after serum addition, cells were irradiated with UV and then incubated at an indicated time (B) or 30 min (C). Experimental procedures are summarized in a schema (A). Scale bar, 10 μm (C). (D) Each Tet-On RPE1 cell line was transfected with Chk1 3′UTR siRNA according to the reverse transfection procedures (Invitrogen). At 4 h after transfection, the medium was replaced with the fresh growing medium containing Dox. At 24 h after transfection, cells were irradiated with UV light at a dose of 20 J/m 2 and then incubated at an indicated time. After treatment, cells were subjected to anti-Myc immunoprecipitation (IP), followed by immunoblotting with the indicated antibody. Experimental procedures are also summarized in a schema (left).

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: Irradiation, Incubation, Transfection, Immunoprecipitation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

doi: 10.1091/mbc.E11-10-0883

Figure Lengend Snippet: Model for Chk1–Ser-280 phosphorylation by p90 RSK.

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Chk1 (clone name, G4) from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti–pan-Akt (40D4), anti-ERK1/2 (3A7), rabbit anti–Akt-pThr-308 (C31E5E), anti–Akt-pSer-473 (D9E), anti-Bad (D24A9), anti–Bad-pSer-112 (40A9), anti–Bad-pSer-136 (D25H8), anti–Chk1-pSer-345 (133D3), anti–ERK1/2-pThr-202/pTyr-204 (D13.14.4E), anti– MAPK-activated protein kinase-2–pThr-334, anti–p90 RSK1/RSK2/RSK3 (32D7), and anti–RSK-pThr-573 from Cell Signaling Technology (Beverly, MA), mouse anti-Chk1 (DCS-310) from Sigma-Aldrich, mouse anti-Myc (4A6) from Millipore (Bedford, MA), and anti–Chk1-pSer-280 from Epitomics (Burlingame, CA).

Techniques: