polyclonal rabbit anti human smc1 igg Search Results


smc1  (Novus Biologicals)
90
Novus Biologicals smc1
Smc1, supplied by Novus Biologicals, 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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smc1  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc smc1
Figure 2 | ARF activation in BRCA2-deficient mouse and human cells is ATM-dependent. (a) MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), in the presence or absence of ATM inhibitor Ku55933. <t>SMC1</t> and tubulin were used as loading controls. (b) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h. Five days after infection, cells were transfected with control or ATM siRNAs. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 and GAPDH were used as loading controls. (c) ATM-deficient GM16666 human cells and ATM-expressing GM16667 cells were transfected with control or BRCA2 siRNAs twice at 3 days interval. Extracts were prepared 6 days after the first transfection and immunoblotted as indicated. GM16667 cells used as control were irradiated (10 Gy) and extracts were prepared 2 h later. SMC1 was used as a loading control. *, nonspecific band.
Smc1, supplied by Cell Signaling Technology Inc, 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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anti smc1 mouse monoclonal  (Bethyl)
96
Bethyl anti smc1 mouse monoclonal
Figure 2 | ARF activation in BRCA2-deficient mouse and human cells is ATM-dependent. (a) MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), in the presence or absence of ATM inhibitor Ku55933. <t>SMC1</t> and tubulin were used as loading controls. (b) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h. Five days after infection, cells were transfected with control or ATM siRNAs. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 and GAPDH were used as loading controls. (c) ATM-deficient GM16666 human cells and ATM-expressing GM16667 cells were transfected with control or BRCA2 siRNAs twice at 3 days interval. Extracts were prepared 6 days after the first transfection and immunoblotted as indicated. GM16667 cells used as control were irradiated (10 Gy) and extracts were prepared 2 h later. SMC1 was used as a loading control. *, nonspecific band.
Anti Smc1 Mouse Monoclonal, supplied by Bethyl, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho smc1  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology phospho smc1
FIG. 1. Chk1 inhibition causes massive phosphorylation of ATR targets in S-phase cells. (A) -H2AX measured at 3 h after Chk1 inhibition by UCN-01 (300 nM) or CEP-3891 (500 nM) or after transfection with Chk1 siRNA (100 nM, 48 h) in U-2-OS cells. Immunofluorescence of cells stained with an antibody to -H2AX is shown. (B) Flow cytometry analysis of cells stained with -H2AX and PI. Numbers indicate the percentages of -H2AX-positive cells. (C) BrdU incorporation in -H2AX-positive cells. A short pulse of BrdU (5 min) was administered to U-2-OS cells at 3 h after treatment with CEP-3891 (500 nM), and cells were then processed for immunofluorescence analysis with antibody to -H2AX, followed by DNase treatment and staining with an antibody to BrdU. (D) Phosphorylation of Chk1, p53, <t>Smc1,</t> and H2AX increase after Chk1 inhibition. Extracts from U-2-OS cells were prepared at 0, 1, 3, and 6 h after treatment with UCN-01 (300 nM) and processed for Western blotting. Mcm7 protein is a loading marker. (E) Phosphorylation of -H2AX, p53, and Smc1 increase after depletion of Chk1 by siRNA transfection. Extracts from U-2-OS cells were prepared at 48 h after transfection with control siRNA () or Chk1 siRNA () and processed for Western blotting.
Phospho Smc1, supplied by Santa Cruz Biotechnology, 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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anti beta galactosidase  (Novus Biologicals)
92
Novus Biologicals anti beta galactosidase
FIG. 1. Chk1 inhibition causes massive phosphorylation of ATR targets in S-phase cells. (A) -H2AX measured at 3 h after Chk1 inhibition by UCN-01 (300 nM) or CEP-3891 (500 nM) or after transfection with Chk1 siRNA (100 nM, 48 h) in U-2-OS cells. Immunofluorescence of cells stained with an antibody to -H2AX is shown. (B) Flow cytometry analysis of cells stained with -H2AX and PI. Numbers indicate the percentages of -H2AX-positive cells. (C) BrdU incorporation in -H2AX-positive cells. A short pulse of BrdU (5 min) was administered to U-2-OS cells at 3 h after treatment with CEP-3891 (500 nM), and cells were then processed for immunofluorescence analysis with antibody to -H2AX, followed by DNase treatment and staining with an antibody to BrdU. (D) Phosphorylation of Chk1, p53, <t>Smc1,</t> and H2AX increase after Chk1 inhibition. Extracts from U-2-OS cells were prepared at 0, 1, 3, and 6 h after treatment with UCN-01 (300 nM) and processed for Western blotting. Mcm7 protein is a loading marker. (E) Phosphorylation of -H2AX, p53, and Smc1 increase after depletion of Chk1 by siRNA transfection. Extracts from U-2-OS cells were prepared at 48 h after transfection with control siRNA () or Chk1 siRNA () and processed for Western blotting.
Anti Beta Galactosidase, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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smc1  (Bethyl)
94
Bethyl smc1
(A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to <t>SMC1</t> intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.
Smc1, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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smc3 antibody nb100 207 novus biologicals inc smc1 antibody  (Novus Biologicals)
94
Novus Biologicals smc3 antibody nb100 207 novus biologicals inc smc1 antibody
(A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to <t>SMC1</t> intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.
Smc3 Antibody Nb100 207 Novus Biologicals Inc Smc1 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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smc3 antibody nb100 207 novus biologicals inc smc1 antibody - by Bioz Stars, 2026-03
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mouse anti phospho smc1 s966  (Bethyl)
93
Bethyl mouse anti phospho smc1 s966
(A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to <t>SMC1</t> intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.
Mouse Anti Phospho Smc1 S966, supplied by Bethyl, 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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anti ps957 smc1  (Novus Biologicals)
90
Novus Biologicals anti ps957 smc1
(A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to <t>SMC1</t> intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.
Anti Ps957 Smc1, supplied by Novus Biologicals, 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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smc1 ps966  (Novus Biologicals)
90
Novus Biologicals smc1 ps966
(A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to <t>SMC1</t> intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.
Smc1 Ps966, supplied by Novus Biologicals, 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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Image Search Results


Figure 2 | ARF activation in BRCA2-deficient mouse and human cells is ATM-dependent. (a) MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), in the presence or absence of ATM inhibitor Ku55933. SMC1 and tubulin were used as loading controls. (b) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h. Five days after infection, cells were transfected with control or ATM siRNAs. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 and GAPDH were used as loading controls. (c) ATM-deficient GM16666 human cells and ATM-expressing GM16667 cells were transfected with control or BRCA2 siRNAs twice at 3 days interval. Extracts were prepared 6 days after the first transfection and immunoblotted as indicated. GM16667 cells used as control were irradiated (10 Gy) and extracts were prepared 2 h later. SMC1 was used as a loading control. *, nonspecific band.

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 2 | ARF activation in BRCA2-deficient mouse and human cells is ATM-dependent. (a) MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), in the presence or absence of ATM inhibitor Ku55933. SMC1 and tubulin were used as loading controls. (b) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h. Five days after infection, cells were transfected with control or ATM siRNAs. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 and GAPDH were used as loading controls. (c) ATM-deficient GM16666 human cells and ATM-expressing GM16667 cells were transfected with control or BRCA2 siRNAs twice at 3 days interval. Extracts were prepared 6 days after the first transfection and immunoblotted as indicated. GM16667 cells used as control were irradiated (10 Gy) and extracts were prepared 2 h later. SMC1 was used as a loading control. *, nonspecific band.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Activation Assay, Infection, Expressing, Control, Plasmid Preparation, Selection, Transfection, Western Blot, Irradiation

Figure 4 | ATR induces ARF expression in response to HR deficiency or oncogenic stress. (a) Human H1299 cells were infected with lentiviruses expressing indicated shRNAs, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 was used as a loading control. (b) MEFs established from p53 / embryos were infected three times with control or RAD51 shRNAs, in the presence or absence of ATM or ATR chemical inhibitors (ATMi, ATRi). Extracts prepared from cells collected 6 days after the first infection were immunoblotted as indicated. Tubulin was used as a loading control. (c) MEFs established from p53 / embryos were infected with control or K-RAS-expressing retroviruses, in the presence of control or ATR shRNAs. Extracts prepared from cells collected 6 days after the first infection were immunoblotted as indicated. ERK, SMC1 and H3 were used as loading controls. (d) Quantification of replication fork speed and tract length using single DNA fibre analysis in cells treated as in c. Error bars represent s.d. of two independent experiments. The statistical significance of the observed reduction in replication tract length induced by K-RAS expression was evaluated using an unpaired two-tailed t-test. Representative images of ongoing replication forks identified with CldU and IdU staining are also shown. (e) Schematic diagram of the DNA damage response to replicative stress, leading to ARF accumulation.

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 4 | ATR induces ARF expression in response to HR deficiency or oncogenic stress. (a) Human H1299 cells were infected with lentiviruses expressing indicated shRNAs, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 was used as a loading control. (b) MEFs established from p53 / embryos were infected three times with control or RAD51 shRNAs, in the presence or absence of ATM or ATR chemical inhibitors (ATMi, ATRi). Extracts prepared from cells collected 6 days after the first infection were immunoblotted as indicated. Tubulin was used as a loading control. (c) MEFs established from p53 / embryos were infected with control or K-RAS-expressing retroviruses, in the presence of control or ATR shRNAs. Extracts prepared from cells collected 6 days after the first infection were immunoblotted as indicated. ERK, SMC1 and H3 were used as loading controls. (d) Quantification of replication fork speed and tract length using single DNA fibre analysis in cells treated as in c. Error bars represent s.d. of two independent experiments. The statistical significance of the observed reduction in replication tract length induced by K-RAS expression was evaluated using an unpaired two-tailed t-test. Representative images of ongoing replication forks identified with CldU and IdU staining are also shown. (e) Schematic diagram of the DNA damage response to replicative stress, leading to ARF accumulation.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Expressing, Infection, Selection, Western Blot, Control, Two Tailed Test, Staining

Figure 3 | MRE11-dependent ssDNA formation triggers ARF induction in BRCA2- and RAD51-deficient cells. (a) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h, in the presence or absence of MRE11 inhibitor mirin. Single-stranded DNA in these cells was quantified using FACS analysis of BrdU immunofluorescence detected under non-denaturing conditions. (b) Cell extracts from cells treated as in a were prepared 6 days after infection and analysed using western blotting. SMC1 and H2AX were used as loading controls. (c) MEFs established from p53 / embryos were treated with GFP control or RAD51 shRNAs followed by selection with puromycin for 48 h, in the presence or absence of MRE11 inhibitor mirin. Extracts were prepared from cells collected 6 days after infection and immunoblotted as indicated. Histone H2AX was used as a loading control.

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 3 | MRE11-dependent ssDNA formation triggers ARF induction in BRCA2- and RAD51-deficient cells. (a) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h, in the presence or absence of MRE11 inhibitor mirin. Single-stranded DNA in these cells was quantified using FACS analysis of BrdU immunofluorescence detected under non-denaturing conditions. (b) Cell extracts from cells treated as in a were prepared 6 days after infection and analysed using western blotting. SMC1 and H2AX were used as loading controls. (c) MEFs established from p53 / embryos were treated with GFP control or RAD51 shRNAs followed by selection with puromycin for 48 h, in the presence or absence of MRE11 inhibitor mirin. Extracts were prepared from cells collected 6 days after infection and immunoblotted as indicated. Histone H2AX was used as a loading control.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Infection, Expressing, Control, Selection, Western Blot

Figure 5 | ARF inhibition rescues senescence and proliferation arrest induced by BRCA2 or RAD51 inactivation in mouse and human cells. (a) Early- passage MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), together with retroviruses expressing ARF or GFP control shRNA, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after the first infection and were analysed using western blotting. SMC1 was used as a loading control. (b) Quantification of the SA-b-gal staining of cells treated as in a. Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to concomitant Brca2 deletion and ARF depletion was evaluated using an unpaired two-tailed t-test. ARF-1sh and ARF-2sh are two independent shRNAs against mouse ARF. (c) Human MRC5 cells were infected with lentiviruses expressing indicated shRNAs, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1, GAPDH and tubulin were used as loading controls. (d) Quantification of the SA-b-gal staining of cells treated as in c. Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to concomitant BRCA2 and ARF depletion was evaluated using an unpaired two-tailed t-test. (e) Early- passage MEFs established from wild-type, p53 / or Arf / embryos were treated with GFP control or RAD51 shRNAs. Extracts were prepared from cells collected 6 days after infection and immunoblotted as indicated. Tubulin was used as a loading control. (f) Quantification of the SA-b-gal staining of cells treated as in (e). Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to RAD51 depletion in the three types of MEFs was evaluated using an unpaired two-tailed t-test. ARF-1sh and ARF-2sh are two independent shRNAs against human ARF. (g) Quantification of the DSBs frequency in metaphase spreads from cells treated as in e. (h) Cell proliferation assays for cells treated as in e. Error bars represent s.d. of three independent experiments.

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 5 | ARF inhibition rescues senescence and proliferation arrest induced by BRCA2 or RAD51 inactivation in mouse and human cells. (a) Early- passage MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), together with retroviruses expressing ARF or GFP control shRNA, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after the first infection and were analysed using western blotting. SMC1 was used as a loading control. (b) Quantification of the SA-b-gal staining of cells treated as in a. Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to concomitant Brca2 deletion and ARF depletion was evaluated using an unpaired two-tailed t-test. ARF-1sh and ARF-2sh are two independent shRNAs against mouse ARF. (c) Human MRC5 cells were infected with lentiviruses expressing indicated shRNAs, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1, GAPDH and tubulin were used as loading controls. (d) Quantification of the SA-b-gal staining of cells treated as in c. Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to concomitant BRCA2 and ARF depletion was evaluated using an unpaired two-tailed t-test. (e) Early- passage MEFs established from wild-type, p53 / or Arf / embryos were treated with GFP control or RAD51 shRNAs. Extracts were prepared from cells collected 6 days after infection and immunoblotted as indicated. Tubulin was used as a loading control. (f) Quantification of the SA-b-gal staining of cells treated as in (e). Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to RAD51 depletion in the three types of MEFs was evaluated using an unpaired two-tailed t-test. ARF-1sh and ARF-2sh are two independent shRNAs against human ARF. (g) Quantification of the DSBs frequency in metaphase spreads from cells treated as in e. (h) Cell proliferation assays for cells treated as in e. Error bars represent s.d. of three independent experiments.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Inhibition, Infection, Expressing, Control, Plasmid Preparation, shRNA, Selection, Western Blot, Staining, Two Tailed Test

FIG. 1. Chk1 inhibition causes massive phosphorylation of ATR targets in S-phase cells. (A) -H2AX measured at 3 h after Chk1 inhibition by UCN-01 (300 nM) or CEP-3891 (500 nM) or after transfection with Chk1 siRNA (100 nM, 48 h) in U-2-OS cells. Immunofluorescence of cells stained with an antibody to -H2AX is shown. (B) Flow cytometry analysis of cells stained with -H2AX and PI. Numbers indicate the percentages of -H2AX-positive cells. (C) BrdU incorporation in -H2AX-positive cells. A short pulse of BrdU (5 min) was administered to U-2-OS cells at 3 h after treatment with CEP-3891 (500 nM), and cells were then processed for immunofluorescence analysis with antibody to -H2AX, followed by DNase treatment and staining with an antibody to BrdU. (D) Phosphorylation of Chk1, p53, Smc1, and H2AX increase after Chk1 inhibition. Extracts from U-2-OS cells were prepared at 0, 1, 3, and 6 h after treatment with UCN-01 (300 nM) and processed for Western blotting. Mcm7 protein is a loading marker. (E) Phosphorylation of -H2AX, p53, and Smc1 increase after depletion of Chk1 by siRNA transfection. Extracts from U-2-OS cells were prepared at 48 h after transfection with control siRNA () or Chk1 siRNA () and processed for Western blotting.

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Human Chk1 Causes Increased Initiation of DNA Replication, Phosphorylation of ATR Targets, and DNA Breakage

doi: 10.1128/mcb.25.9.3553-3562.2005

Figure Lengend Snippet: FIG. 1. Chk1 inhibition causes massive phosphorylation of ATR targets in S-phase cells. (A) -H2AX measured at 3 h after Chk1 inhibition by UCN-01 (300 nM) or CEP-3891 (500 nM) or after transfection with Chk1 siRNA (100 nM, 48 h) in U-2-OS cells. Immunofluorescence of cells stained with an antibody to -H2AX is shown. (B) Flow cytometry analysis of cells stained with -H2AX and PI. Numbers indicate the percentages of -H2AX-positive cells. (C) BrdU incorporation in -H2AX-positive cells. A short pulse of BrdU (5 min) was administered to U-2-OS cells at 3 h after treatment with CEP-3891 (500 nM), and cells were then processed for immunofluorescence analysis with antibody to -H2AX, followed by DNase treatment and staining with an antibody to BrdU. (D) Phosphorylation of Chk1, p53, Smc1, and H2AX increase after Chk1 inhibition. Extracts from U-2-OS cells were prepared at 0, 1, 3, and 6 h after treatment with UCN-01 (300 nM) and processed for Western blotting. Mcm7 protein is a loading marker. (E) Phosphorylation of -H2AX, p53, and Smc1 increase after depletion of Chk1 by siRNA transfection. Extracts from U-2-OS cells were prepared at 48 h after transfection with control siRNA () or Chk1 siRNA () and processed for Western blotting.

Article Snippet: Goat antibody to ATR (sc-1887) and rabbit antibodies to Cdc45 (sc-20685) and Cdk2 (sc-163) were purchased from Santa Cruz, and rabbit antibody to phospho-SMC1 (Ser 966) was purchased from Ab-cam.

Techniques: Inhibition, Phospho-proteomics, Transfection, Staining, Flow Cytometry, BrdU Incorporation Assay, Western Blot, Marker, Control

(A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to SMC1 intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.

Journal: Science signaling

Article Title: Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs

doi: 10.1126/scisignal.aap8112

Figure Lengend Snippet: (A to C) Graphs show the estimated mass of proteins annotated with the KEGG pathway terms “oxidative phosphorylation” (OxPhos) (A), “TCA cycle” (B), or “glycolysis” (C) per cell in IL-2-maintained and IL-2-deprived CTLs. Estimated mass per protein was determined using estimated copy numbers calculated from the proteomic data using the histone ruler method. The bar charts in (D)-(E), (G) and (I)-(L) show estimated copy numbers per cell for GLUT1 (SLC2A1) (D), GLUT3 (SLC2A3) (E), HIF1β (G), TIM-3 (I), NFIL3 (J), L-selectin (CD62L) (K) and PHD2 (L). (F) Glucose uptake in IL-2-maintained and IL-2-deprived CTLs was measured using radiolabeled 2-deoxyglucose. Data were normalized to glucose uptake in IL-2 maintained CTL. (H) HIF1α abundance in CTL in the presence and absence of IL-2 was measured by immunoblot. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to SMC1 intensity and HIF1α protein abundance in IL-2 maintained CTL. In bar charts, individual data points from the three biological replicates are shown and color matched, with the bar showing the mean and the errors bars showing standard deviation of the experiments. P values shown are two-tailed one sample Student’s t-test performed on the ratios of no IL-2/IL-2.

Article Snippet: Membranes were incubated with the following primary antibodies: STAT5A/B (CST #9363), STAT5A/B pTyr 694 /pTyr 699 (CST, #9359), NFIL3 (CST #14312), granzyme B (CST #4275), perforin-1 (CST #3693), HIF-1α (R&D Systems, clone 241809, cat. MAB15361), SMC1 (Bethyl labs, cat. A300-005A), S6 pSer 235 /pSer 236 (CST #2211), S6 (CST #2217), S6K pThr 389 (CST #9205), S6K (CST #9202), AKT pThr 308 (CST #4056), AKT pSer 473 (CST #4058), AKT (CST #9272), FOXO1/3A pThr 24 /pThr 32 (CST #9464), and FOXO1 (CST #9454).

Techniques: Western Blot, Standard Deviation, Two Tailed Test

(A) Schematic representation of selected molecules regulated by Tofacitinib. (B) Volcano plot showing the protein copy number ratio in IL-2-maintained CTLs treated with 100 nM Tofacitinib for 24 hours compared to IL-2-maintained CTLs, significantly regulated proteins are shown in dark grey and nutrient transporters are highlighted in red. Estimated copy numbers per cell are shown for SESTRIN2 (C), GLUT1 (D) and GLUT3 (E). (F) Uptake of a radiolabelled glucose analog shown relative to IL-2. (G) Lactate output in IL-2-maintained and Tofacitinib-treated CTLs. Estimated copy numbers per cell for perforin (H) and NFIL3 (I). (J) Western blot analysis of HIF1α abundance in response to 100 nM Tofacitinib. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to SMC1 intensity and HIF1α protein abundance in IL-2 maintained CTL. The data show, or are representative of, three biological replicates. In bar charts, data points from biological replicates are color matched, the bar shows the mean and the errors bars show standard deviation. P values shown are two-tailed one sample Student’s t-test performed on the ratios of IL-2+Tofacitinib/IL-2.

Journal: Science signaling

Article Title: Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs

doi: 10.1126/scisignal.aap8112

Figure Lengend Snippet: (A) Schematic representation of selected molecules regulated by Tofacitinib. (B) Volcano plot showing the protein copy number ratio in IL-2-maintained CTLs treated with 100 nM Tofacitinib for 24 hours compared to IL-2-maintained CTLs, significantly regulated proteins are shown in dark grey and nutrient transporters are highlighted in red. Estimated copy numbers per cell are shown for SESTRIN2 (C), GLUT1 (D) and GLUT3 (E). (F) Uptake of a radiolabelled glucose analog shown relative to IL-2. (G) Lactate output in IL-2-maintained and Tofacitinib-treated CTLs. Estimated copy numbers per cell for perforin (H) and NFIL3 (I). (J) Western blot analysis of HIF1α abundance in response to 100 nM Tofacitinib. Quantification of HIF1α detected by Western blot is shown alongside, with data normalized to SMC1 intensity and HIF1α protein abundance in IL-2 maintained CTL. The data show, or are representative of, three biological replicates. In bar charts, data points from biological replicates are color matched, the bar shows the mean and the errors bars show standard deviation. P values shown are two-tailed one sample Student’s t-test performed on the ratios of IL-2+Tofacitinib/IL-2.

Article Snippet: Membranes were incubated with the following primary antibodies: STAT5A/B (CST #9363), STAT5A/B pTyr 694 /pTyr 699 (CST, #9359), NFIL3 (CST #14312), granzyme B (CST #4275), perforin-1 (CST #3693), HIF-1α (R&D Systems, clone 241809, cat. MAB15361), SMC1 (Bethyl labs, cat. A300-005A), S6 pSer 235 /pSer 236 (CST #2211), S6 (CST #2217), S6K pThr 389 (CST #9205), S6K (CST #9202), AKT pThr 308 (CST #4056), AKT pSer 473 (CST #4058), AKT (CST #9272), FOXO1/3A pThr 24 /pThr 32 (CST #9464), and FOXO1 (CST #9454).

Techniques: Western Blot, Standard Deviation, Two Tailed Test

(A) Average frequency of protein copy numbers per cell calculated from the proteomic datasets: The bin that contains NFIL3 is indicated in black. (B) Western blot analysis of NFIL3 abundance in CTLs following IL-2 deprivation or treatment with 100 nM Tofacitinib. Quantification is shown alongside as data normalized to SMC1 intensity and NFIL3 protein abundance in IL-2 maintained CTL. (C) Abundance of Nfil3 mRNA in CTLs. Abundance of NFIL3 protein (D) and mRNA (E) in naïve CD8+ T cells, TCR-activated CD8+ T cells, and CTLs activated for 48 hours and then maintained in IL-2 for 1 day, 3 days, or 5 days. (F) Analysis of NFIL3 protein abundance in HIF1α expressing (Hif1a+/+) and HIF1α deficient (Hif1a-/-) CTLs maintained in normoxia (21% O2) or switched into hypoxic (1% O2) conditions for eight hours. Quantification of NFIL3 protein is shown alongside. Relative abundance of Nfil3 (G) and GLUT1 (Slc2a1) (H) mRNA in CTLs maintained in normoxia or switched into 1% O2 for four hours. Abundance of CD62L (Sell) mRNA (I) and cell-surface protein abundance (J) in IL-2-maintained Nfil3+/+ and Nfil3-/- CTL. In (J), the percentage of CD8+ CD62L high CTLs are shown alongside. Abundance of perforin (Prf1) mRNA (K) and protein (L) in Nfil3+/+ and Nfil3-/- CTLs. In (L) quantification of NFIL3, normalized to SMC1 and perforin protein abundance in IL-2 maintained CTL, detected by Western blot is shown alongside. In (C), (G), and (H) the qPCR data were normalized to Tbp, and mRNA abundance in IL-2 maintained CTL in normoxia. In (E) qPCR data were normalized to Tbp and Cd8 and all are shown relative to day 5 IL-2-maintained CTLs in normoxia. In (I) and (K), mRNA was normalized to Hprt, and data are shown relative to the abundance in Nfil3+/+ CTLs. Data in (A)-(E) and (G)-(H) data show, or are representative of, three biological replicates; in (F) data are representative of six Hif1a+/+ and five Hif1a-/- biological replicates, four independent experiments; in (I)-(L) data are representative of four Nfil3+/+ and three Nfil3-/- biological replicates. Bar charts show data from individual biological replicates, color matched where appropriate, the bar shows the mean and the errors bars show standard deviation. P values shown in (B)-(H) are two-tailed one sample Student’s t-tests and in (I)-(L) are two-tailed unpaired unequal variance Student’s t-tests.

Journal: Science signaling

Article Title: Interleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs

doi: 10.1126/scisignal.aap8112

Figure Lengend Snippet: (A) Average frequency of protein copy numbers per cell calculated from the proteomic datasets: The bin that contains NFIL3 is indicated in black. (B) Western blot analysis of NFIL3 abundance in CTLs following IL-2 deprivation or treatment with 100 nM Tofacitinib. Quantification is shown alongside as data normalized to SMC1 intensity and NFIL3 protein abundance in IL-2 maintained CTL. (C) Abundance of Nfil3 mRNA in CTLs. Abundance of NFIL3 protein (D) and mRNA (E) in naïve CD8+ T cells, TCR-activated CD8+ T cells, and CTLs activated for 48 hours and then maintained in IL-2 for 1 day, 3 days, or 5 days. (F) Analysis of NFIL3 protein abundance in HIF1α expressing (Hif1a+/+) and HIF1α deficient (Hif1a-/-) CTLs maintained in normoxia (21% O2) or switched into hypoxic (1% O2) conditions for eight hours. Quantification of NFIL3 protein is shown alongside. Relative abundance of Nfil3 (G) and GLUT1 (Slc2a1) (H) mRNA in CTLs maintained in normoxia or switched into 1% O2 for four hours. Abundance of CD62L (Sell) mRNA (I) and cell-surface protein abundance (J) in IL-2-maintained Nfil3+/+ and Nfil3-/- CTL. In (J), the percentage of CD8+ CD62L high CTLs are shown alongside. Abundance of perforin (Prf1) mRNA (K) and protein (L) in Nfil3+/+ and Nfil3-/- CTLs. In (L) quantification of NFIL3, normalized to SMC1 and perforin protein abundance in IL-2 maintained CTL, detected by Western blot is shown alongside. In (C), (G), and (H) the qPCR data were normalized to Tbp, and mRNA abundance in IL-2 maintained CTL in normoxia. In (E) qPCR data were normalized to Tbp and Cd8 and all are shown relative to day 5 IL-2-maintained CTLs in normoxia. In (I) and (K), mRNA was normalized to Hprt, and data are shown relative to the abundance in Nfil3+/+ CTLs. Data in (A)-(E) and (G)-(H) data show, or are representative of, three biological replicates; in (F) data are representative of six Hif1a+/+ and five Hif1a-/- biological replicates, four independent experiments; in (I)-(L) data are representative of four Nfil3+/+ and three Nfil3-/- biological replicates. Bar charts show data from individual biological replicates, color matched where appropriate, the bar shows the mean and the errors bars show standard deviation. P values shown in (B)-(H) are two-tailed one sample Student’s t-tests and in (I)-(L) are two-tailed unpaired unequal variance Student’s t-tests.

Article Snippet: Membranes were incubated with the following primary antibodies: STAT5A/B (CST #9363), STAT5A/B pTyr 694 /pTyr 699 (CST, #9359), NFIL3 (CST #14312), granzyme B (CST #4275), perforin-1 (CST #3693), HIF-1α (R&D Systems, clone 241809, cat. MAB15361), SMC1 (Bethyl labs, cat. A300-005A), S6 pSer 235 /pSer 236 (CST #2211), S6 (CST #2217), S6K pThr 389 (CST #9205), S6K (CST #9202), AKT pThr 308 (CST #4056), AKT pSer 473 (CST #4058), AKT (CST #9272), FOXO1/3A pThr 24 /pThr 32 (CST #9464), and FOXO1 (CST #9454).

Techniques: Western Blot, Expressing, Standard Deviation, Two Tailed Test