other:
SDS Page:Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo.
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies.
Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 °C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. The membrane was incubated with chemiluminescence horseradish peroxidase (HRP) substrate, the signal was captured on the Chemidoc system, and quantification was performed using ImageJ software (2.16.0).
Membrane:Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo.
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies.
Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 °C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. The membrane was incubated with chemiluminescence horseradish peroxidase (HRP) substrate, the signal was captured on the Chemidoc system, and quantification was performed using ImageJ software (2.16.0).
Binding Assay:Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo.
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies.
Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 °C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. The membrane was incubated with chemiluminescence horseradish peroxidase (HRP) substrate, the signal was captured on the Chemidoc system, and quantification was performed using ImageJ software (2.16.0).
Incubation:Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo.
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 ◦C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies.
Article Title: Novel Cell Cycle Inhibitors Decrease Primary and Metastatic Breast Cancer Growth In Vivo
Article Snippet: .. Equal amounts of protein from each sample were subjected to 8% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 4 °C at 100 V. Then, the PVDF membrane was blocked with 5% non-fat milk or 5% BSA in order to avoid non-specific binding, followed by incubation with target primary antibodies (p-Rpb1 CTD (Ser2/5) (4735 CS), Rpb1 CTD (2629 CST), p-Rb (Ser807/811) (8516 CST), Rb (4H1) (mouse mAb 9309 CST), and β-actin A3854 (Sigma, Merck KGaA, St. Louis, MO, USA)) and their respective HRP-conjugated secondary antibodies. .. The membrane was incubated with chemiluminescence horseradish peroxidase (HRP) substrate, the signal was captured on the Chemidoc system, and quantification was performed using ImageJ software (2.16.0).
Ubiquitin Proteomics:Article Title: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry
Article Snippet: .. The following antibodies were used in this study; CDH1 (FZR1) antibody (Abcam, Ab217038 , 1:1,000, IP: 2 μg; Santa Cruz, sc-56312, IB: 1:800; Sigma-Aldrich, CC43-100UG, 1:2,000), mTOR antibody (CST, 2972, 1:1,000), p-mTOR (CST, 2971, 1:1,000), mCherry (Abcam, ab167453, 1:1,000), geminin (CST, 5165, 1:1,000), cyclin D1 (Thermo Fisher Scientific, MA5 14512, 1:750), p-Rb (Ser807/811) (CST, 8516, 1:1,000), Rb (CST, 9309, 1:2,000), vinculin (Sigma-Aldrich, V9131, 1:10,000), Ki-67 (Abcam, ab8191, 1:1,000), p21 (BD Biosciences, 556430, 1:1,000), p27 (CST, 3686, 1:1,000), His tag (Santa Cruz, sc-8036, 1:800), anti-DDK/Flag (Sigma-Aldrich, F3165, 1:1,000), p-S6 kinase (CST, 9205, 1:1,000), APC2 (CST, 12301, 1:1,000), APC6 (CST, 9499, 1:1,000), APC11 (CST, 14090, 1:1,000), S6 kinase (CST, 9202, 1:1,000), p-4EBP1 (CST, 2855, 1:1000), cyclin A2 (Santa Cruz, sc-271682, 1:500), EMI1 (Santa Cruz, sc-365212, 1:500), cyclin F (Santa Cruz, sc-515207, 1:500), βTrCP (CST, 4394, 1:1,000), pSer/Thr/Tyr (Thermo Fisher Scientific, 61-8300, 1:1,000), pThr (Abcam, ab9337, 1:500), pTyr (Abcam, ab10321, 1:1,000), pan anti-phospho-serine/threonine antibody (Phospho Solutions, PP2551; 1:2,000), HA (Santa Cruz, sc-7392, 1:800, IP: 2 μg; and CST, 3724, 1:1,000), GST (Santa Cruz, sc-138, 1:750), PFKFB3 (MBS, 9604769, IB: 1:750, IP: 2 μg), PFKFB3 (Abcam, AB181861-1001, 1:4,000), PFKFB2 (CST, 13029, 1:1,000), PFKFB1 (Abcam, ab155564, 1:1,000), β-actin (Abcam, ab6276, 1:2,000), RPTOR (CST, 2280, 1:1,000), RICTOR (CST, 2114, 1:1,000), mLST8 (CST, 3274, 1:1,000), ubiquitin (Santa Cruz, sc-8017, 1:800), TSC1 (CST, 6935, 1:1,000), NPRL2 (CST, 37344, 1:1,000), histone H1 (Abcam, 11079, 1:1,000), EGFR (Santa Cruz, sc-373746, 1:800), AKT (CST, 9272, 1:1,000), p-AKT (CST, 4060, 1:1,000), mouse anti-goat IgG-HRP (Santa Cruz, sc-2354, 1:10,000), anti-rabbit IgG, HRP-linked antibody (CST, 7074, 1:10,000), mouse anti-rabbit secondary-HRP (Santa Cruz, sc-2357, 1:2,500) or recombinant anti-mouse (Santa Cruz, sc-516102, 1:2,500), anti-mouse IgG, HRP-linked antibody (CST, 7076, 1:10,000), normal rabbit IgG (CST, 2729, IP: 2 μg), normal mouse IgG (Santa Cruz, sc-2025, IP: 2 μg). ..
Article Title: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry.
Article Snippet: .. The following antibodies were used in this study; CDH1 (FZR1) antibody (Abcam, Ab217038, 1:1,000, IP: 2 μg; Santa Cruz, sc-56312, IB: 1:800; Sigma-Aldrich, CC43-100UG, 1:2,000), mTOR antibody (CST, 2972, 1:1,000), p-mTOR (CST, 2971, 1:1,000), mCherry (Abcam, ab167453, 1:1,000), geminin (CST, 5165, 1:1,000), cyclin D1 (Thermo Fisher Scientific, MA5 14512, 1:750), p-Rb (Ser807/811) (CST, 8516, 1:1,000), Rb (CST, 9309, 1:2,000), vinculin (Sigma-Aldrich, V9131, 1:10,000), Ki-67 (Abcam, ab8191, 1:1,000), p21 (BD Biosciences, 556430, 1:1,000), p27 (CST, 3686, 1:1,000), His tag (Santa Cruz, sc-8036, 1:800), anti-DDK/ Flag (Sigma-Aldrich, F3165, 1:1,000), p-S6 kinase (CST, 9205, 1:1,000), APC2 (CST, 12301, 1:1,000), APC6 (CST, 9499, 1:1,000), APC11 (CST, 14090, 1:1,000), S6 kinase (CST, 9202, 1:1,000), p-4EBP1 (CST, 2855, 1:1000), cyclin A2 (Santa Cruz, sc-271682, 1:500), EMI1 (Santa Cruz, sc-365212, 1:500), cyclin F (Santa Cruz, sc-515207, 1:500), βTrCP (CST, 4394, 1:1,000), pSer/Thr/Tyr (Thermo Fisher Scientific, 61-8300, 1:1,000), pThr (Abcam, ab9337, 1:500), pTyr (Abcam, ab10321, 1:1,000), pan anti-phospho-serine/threonine antibody (Phospho Solutions, PP2551; 1:2,000), HA (Santa Cruz, sc-7392, 1:800, IP: 2 μg; and CST, 3724, 1:1,000), GST (Santa Cruz, sc-138, 1:750), PFKFB3 (MBS, 9604769, IB: 1:750, IP: 2 μg), PFKFB3 (Abcam, AB181861-1001, 1:4,000), PFKFB2 (CST, 13029, 1:1,000), PFKFB1 (Abcam, ab155564, 1:1,000), β-actin (Abcam, ab6276, 1:2,000), RPTOR (CST, 2280, 1:1,000), RICTOR (CST, 2114, 1:1,000), mLST8 (CST, 3274, 1:1,000), ubiquitin (Santa Cruz, sc-8017, 1:800), TSC1 (CST, 6935, 1:1,000), NPRL2 (CST, 37344, 1:1,000), histone H1 (Abcam, 11079, 1:1,000), EGFR (Santa Cruz, sc-373746, 1:800), AKT (CST, 9272, 1:1,000), p-AKT (CST, 4060, 1:1,000), mouse anti-goat IgG-HRP (Santa Cruz, sc-2354, 1:10,000), anti-rabbit IgG, HRP-linked antibody (CST, 7074, 1:10,000), mouse anti-rabbit secondary-HRP (Santa Cruz, sc-2357, 1:2,500) or recombinant anti-mouse (Santa Cruz, sc-516102, 1:2,500), anti-mouse IgG, HRP-linked antibody (CST, 7076, 1:10,000), normal rabbit IgG (CST, 2729, IP: 2 μg), normal mouse IgG (Santa Cruz, sc-2025, IP: 2 μg). siRNA transfection The indicated cells were transfected using Dharmafect 1 (Horizon) according to the manufacturer’s instructions. .. The following siRNAs were used: On-Target plus control siRNA (nontargeting, Dharmacon), On-Target plus pooled set of four siRNAs for CCNA2 (M-003205-020005), FZR1 (L-004086-00-0005), EMI1 (M-012434-01-0005), CCNF (custom, 5′-GCACCCGGUUUAUCAGUAAUUUU-3′), BTRC (encoding βTrCP) (L-003463-00-0005 and L-003490-00-0005), PFKFB3 (L-006763-00-0005), TSC1 (L-003028-00-0005), NPRL2 (L-01564500-0005), CDH1 3′ UTR (Qiagen, SI04955265|S1 (1027417) at final concentrations of 20 nM unless noted.
Recombinant:Article Title: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry
Article Snippet: .. The following antibodies were used in this study; CDH1 (FZR1) antibody (Abcam, Ab217038 , 1:1,000, IP: 2 μg; Santa Cruz, sc-56312, IB: 1:800; Sigma-Aldrich, CC43-100UG, 1:2,000), mTOR antibody (CST, 2972, 1:1,000), p-mTOR (CST, 2971, 1:1,000), mCherry (Abcam, ab167453, 1:1,000), geminin (CST, 5165, 1:1,000), cyclin D1 (Thermo Fisher Scientific, MA5 14512, 1:750), p-Rb (Ser807/811) (CST, 8516, 1:1,000), Rb (CST, 9309, 1:2,000), vinculin (Sigma-Aldrich, V9131, 1:10,000), Ki-67 (Abcam, ab8191, 1:1,000), p21 (BD Biosciences, 556430, 1:1,000), p27 (CST, 3686, 1:1,000), His tag (Santa Cruz, sc-8036, 1:800), anti-DDK/Flag (Sigma-Aldrich, F3165, 1:1,000), p-S6 kinase (CST, 9205, 1:1,000), APC2 (CST, 12301, 1:1,000), APC6 (CST, 9499, 1:1,000), APC11 (CST, 14090, 1:1,000), S6 kinase (CST, 9202, 1:1,000), p-4EBP1 (CST, 2855, 1:1000), cyclin A2 (Santa Cruz, sc-271682, 1:500), EMI1 (Santa Cruz, sc-365212, 1:500), cyclin F (Santa Cruz, sc-515207, 1:500), βTrCP (CST, 4394, 1:1,000), pSer/Thr/Tyr (Thermo Fisher Scientific, 61-8300, 1:1,000), pThr (Abcam, ab9337, 1:500), pTyr (Abcam, ab10321, 1:1,000), pan anti-phospho-serine/threonine antibody (Phospho Solutions, PP2551; 1:2,000), HA (Santa Cruz, sc-7392, 1:800, IP: 2 μg; and CST, 3724, 1:1,000), GST (Santa Cruz, sc-138, 1:750), PFKFB3 (MBS, 9604769, IB: 1:750, IP: 2 μg), PFKFB3 (Abcam, AB181861-1001, 1:4,000), PFKFB2 (CST, 13029, 1:1,000), PFKFB1 (Abcam, ab155564, 1:1,000), β-actin (Abcam, ab6276, 1:2,000), RPTOR (CST, 2280, 1:1,000), RICTOR (CST, 2114, 1:1,000), mLST8 (CST, 3274, 1:1,000), ubiquitin (Santa Cruz, sc-8017, 1:800), TSC1 (CST, 6935, 1:1,000), NPRL2 (CST, 37344, 1:1,000), histone H1 (Abcam, 11079, 1:1,000), EGFR (Santa Cruz, sc-373746, 1:800), AKT (CST, 9272, 1:1,000), p-AKT (CST, 4060, 1:1,000), mouse anti-goat IgG-HRP (Santa Cruz, sc-2354, 1:10,000), anti-rabbit IgG, HRP-linked antibody (CST, 7074, 1:10,000), mouse anti-rabbit secondary-HRP (Santa Cruz, sc-2357, 1:2,500) or recombinant anti-mouse (Santa Cruz, sc-516102, 1:2,500), anti-mouse IgG, HRP-linked antibody (CST, 7076, 1:10,000), normal rabbit IgG (CST, 2729, IP: 2 μg), normal mouse IgG (Santa Cruz, sc-2025, IP: 2 μg). ..
Article Title: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry.
Article Snippet: .. The following antibodies were used in this study; CDH1 (FZR1) antibody (Abcam, Ab217038, 1:1,000, IP: 2 μg; Santa Cruz, sc-56312, IB: 1:800; Sigma-Aldrich, CC43-100UG, 1:2,000), mTOR antibody (CST, 2972, 1:1,000), p-mTOR (CST, 2971, 1:1,000), mCherry (Abcam, ab167453, 1:1,000), geminin (CST, 5165, 1:1,000), cyclin D1 (Thermo Fisher Scientific, MA5 14512, 1:750), p-Rb (Ser807/811) (CST, 8516, 1:1,000), Rb (CST, 9309, 1:2,000), vinculin (Sigma-Aldrich, V9131, 1:10,000), Ki-67 (Abcam, ab8191, 1:1,000), p21 (BD Biosciences, 556430, 1:1,000), p27 (CST, 3686, 1:1,000), His tag (Santa Cruz, sc-8036, 1:800), anti-DDK/ Flag (Sigma-Aldrich, F3165, 1:1,000), p-S6 kinase (CST, 9205, 1:1,000), APC2 (CST, 12301, 1:1,000), APC6 (CST, 9499, 1:1,000), APC11 (CST, 14090, 1:1,000), S6 kinase (CST, 9202, 1:1,000), p-4EBP1 (CST, 2855, 1:1000), cyclin A2 (Santa Cruz, sc-271682, 1:500), EMI1 (Santa Cruz, sc-365212, 1:500), cyclin F (Santa Cruz, sc-515207, 1:500), βTrCP (CST, 4394, 1:1,000), pSer/Thr/Tyr (Thermo Fisher Scientific, 61-8300, 1:1,000), pThr (Abcam, ab9337, 1:500), pTyr (Abcam, ab10321, 1:1,000), pan anti-phospho-serine/threonine antibody (Phospho Solutions, PP2551; 1:2,000), HA (Santa Cruz, sc-7392, 1:800, IP: 2 μg; and CST, 3724, 1:1,000), GST (Santa Cruz, sc-138, 1:750), PFKFB3 (MBS, 9604769, IB: 1:750, IP: 2 μg), PFKFB3 (Abcam, AB181861-1001, 1:4,000), PFKFB2 (CST, 13029, 1:1,000), PFKFB1 (Abcam, ab155564, 1:1,000), β-actin (Abcam, ab6276, 1:2,000), RPTOR (CST, 2280, 1:1,000), RICTOR (CST, 2114, 1:1,000), mLST8 (CST, 3274, 1:1,000), ubiquitin (Santa Cruz, sc-8017, 1:800), TSC1 (CST, 6935, 1:1,000), NPRL2 (CST, 37344, 1:1,000), histone H1 (Abcam, 11079, 1:1,000), EGFR (Santa Cruz, sc-373746, 1:800), AKT (CST, 9272, 1:1,000), p-AKT (CST, 4060, 1:1,000), mouse anti-goat IgG-HRP (Santa Cruz, sc-2354, 1:10,000), anti-rabbit IgG, HRP-linked antibody (CST, 7074, 1:10,000), mouse anti-rabbit secondary-HRP (Santa Cruz, sc-2357, 1:2,500) or recombinant anti-mouse (Santa Cruz, sc-516102, 1:2,500), anti-mouse IgG, HRP-linked antibody (CST, 7076, 1:10,000), normal rabbit IgG (CST, 2729, IP: 2 μg), normal mouse IgG (Santa Cruz, sc-2025, IP: 2 μg). siRNA transfection The indicated cells were transfected using Dharmafect 1 (Horizon) according to the manufacturer’s instructions. .. The following siRNAs were used: On-Target plus control siRNA (nontargeting, Dharmacon), On-Target plus pooled set of four siRNAs for CCNA2 (M-003205-020005), FZR1 (L-004086-00-0005), EMI1 (M-012434-01-0005), CCNF (custom, 5′-GCACCCGGUUUAUCAGUAAUUUU-3′), BTRC (encoding βTrCP) (L-003463-00-0005 and L-003490-00-0005), PFKFB3 (L-006763-00-0005), TSC1 (L-003028-00-0005), NPRL2 (L-01564500-0005), CDH1 3′ UTR (Qiagen, SI04955265|S1 (1027417) at final concentrations of 20 nM unless noted.
Western Blot:Article Title: An inducer of snail hibernation causes quiescence and hibernation-like cardioprotection, through metabolic rewiring and autophagy, in mice hearts
Article Snippet: .. Antibody dilution was 1:1000 for the following immunoblots: p-Rb ser807/811 (Cell Signaling Technology, 8516), Rb (Invitrogen, SY63-03), p-CDK2 Thr160 (Cell Signaling Technology, 2561), t-CDK2 (Abcam, ab32147), β-Actin (Cell Signaling Technology, 3700), t-AMPK (Cell Signaling Technology, 5831), p-AMPK Thr172 (Cell Signaling Technology, 2535), AKT (Cell Signaling Technology, 9272), p-AKT Ser473 (Cell Signaling Technology, 9271), t-S6K1 (Cell Signaling Technology, 2708), and p-S6K1 Thr389 (Cell Signaling Technology, 9205), p-PDHE1α ser232 (Millipore Sigma, AP1063), p-PDHE1α ser300 (Millipore Sigma, AP1064), t-PERK (Cell Signaling Technology, 3192), p-PERK Thr982 (Invitrogen, PA5-40294), ATF6 (Abcam, ab37149), LC3B (Cell Signaling Technology, 2775), cleaved caspase-3 (Cell signaling Technology, 9664), t-mTOR (Cell Signaling Technology, 2983), p-mTOR ser2448 (Cell Signaling Technology, 5536), Ki67 (Abcam, ab16667), p-eIF2α Ser51 (Cell Signaling Technology, 9721), t-eIF2α (Cell Signaling Technology, 5324), Custom p-PDK1 Thr344 (generated by Neobiolab), t-PDK1 (Abcam, ab110025), LDHA (Cell Signaling Technology, 2012), p-JNK Thr183/Tyr185 (Cell Signaling Technology, 9251), t-JNK (Cell Signaling Technology, 3708), α-Tubulin (Cell Signaling Technology, 2144), NHERF-1 (Santa Cruz Biotechnology, sc-271552). .. Antibody dilution was 1:5000 for the following immunoblots: PHLPP (Proteintech, 22789-1-AP), p-PDHE1α ser292 (Abcam, ab92696), t-PDHE1α (Abcam, ab168379), GAPDH (Abcam, ab8245).
Generated:Article Title: An inducer of snail hibernation causes quiescence and hibernation-like cardioprotection, through metabolic rewiring and autophagy, in mice hearts
Article Snippet: .. Antibody dilution was 1:1000 for the following immunoblots: p-Rb ser807/811 (Cell Signaling Technology, 8516), Rb (Invitrogen, SY63-03), p-CDK2 Thr160 (Cell Signaling Technology, 2561), t-CDK2 (Abcam, ab32147), β-Actin (Cell Signaling Technology, 3700), t-AMPK (Cell Signaling Technology, 5831), p-AMPK Thr172 (Cell Signaling Technology, 2535), AKT (Cell Signaling Technology, 9272), p-AKT Ser473 (Cell Signaling Technology, 9271), t-S6K1 (Cell Signaling Technology, 2708), and p-S6K1 Thr389 (Cell Signaling Technology, 9205), p-PDHE1α ser232 (Millipore Sigma, AP1063), p-PDHE1α ser300 (Millipore Sigma, AP1064), t-PERK (Cell Signaling Technology, 3192), p-PERK Thr982 (Invitrogen, PA5-40294), ATF6 (Abcam, ab37149), LC3B (Cell Signaling Technology, 2775), cleaved caspase-3 (Cell signaling Technology, 9664), t-mTOR (Cell Signaling Technology, 2983), p-mTOR ser2448 (Cell Signaling Technology, 5536), Ki67 (Abcam, ab16667), p-eIF2α Ser51 (Cell Signaling Technology, 9721), t-eIF2α (Cell Signaling Technology, 5324), Custom p-PDK1 Thr344 (generated by Neobiolab), t-PDK1 (Abcam, ab110025), LDHA (Cell Signaling Technology, 2012), p-JNK Thr183/Tyr185 (Cell Signaling Technology, 9251), t-JNK (Cell Signaling Technology, 3708), α-Tubulin (Cell Signaling Technology, 2144), NHERF-1 (Santa Cruz Biotechnology, sc-271552). .. Antibody dilution was 1:5000 for the following immunoblots: PHLPP (Proteintech, 22789-1-AP), p-PDHE1α ser292 (Abcam, ab92696), t-PDHE1α (Abcam, ab168379), GAPDH (Abcam, ab8245).
Transfection:Article Title: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry.
Article Snippet: .. The following antibodies were used in this study; CDH1 (FZR1) antibody (Abcam, Ab217038, 1:1,000, IP: 2 μg; Santa Cruz, sc-56312, IB: 1:800; Sigma-Aldrich, CC43-100UG, 1:2,000), mTOR antibody (CST, 2972, 1:1,000), p-mTOR (CST, 2971, 1:1,000), mCherry (Abcam, ab167453, 1:1,000), geminin (CST, 5165, 1:1,000), cyclin D1 (Thermo Fisher Scientific, MA5 14512, 1:750), p-Rb (Ser807/811) (CST, 8516, 1:1,000), Rb (CST, 9309, 1:2,000), vinculin (Sigma-Aldrich, V9131, 1:10,000), Ki-67 (Abcam, ab8191, 1:1,000), p21 (BD Biosciences, 556430, 1:1,000), p27 (CST, 3686, 1:1,000), His tag (Santa Cruz, sc-8036, 1:800), anti-DDK/ Flag (Sigma-Aldrich, F3165, 1:1,000), p-S6 kinase (CST, 9205, 1:1,000), APC2 (CST, 12301, 1:1,000), APC6 (CST, 9499, 1:1,000), APC11 (CST, 14090, 1:1,000), S6 kinase (CST, 9202, 1:1,000), p-4EBP1 (CST, 2855, 1:1000), cyclin A2 (Santa Cruz, sc-271682, 1:500), EMI1 (Santa Cruz, sc-365212, 1:500), cyclin F (Santa Cruz, sc-515207, 1:500), βTrCP (CST, 4394, 1:1,000), pSer/Thr/Tyr (Thermo Fisher Scientific, 61-8300, 1:1,000), pThr (Abcam, ab9337, 1:500), pTyr (Abcam, ab10321, 1:1,000), pan anti-phospho-serine/threonine antibody (Phospho Solutions, PP2551; 1:2,000), HA (Santa Cruz, sc-7392, 1:800, IP: 2 μg; and CST, 3724, 1:1,000), GST (Santa Cruz, sc-138, 1:750), PFKFB3 (MBS, 9604769, IB: 1:750, IP: 2 μg), PFKFB3 (Abcam, AB181861-1001, 1:4,000), PFKFB2 (CST, 13029, 1:1,000), PFKFB1 (Abcam, ab155564, 1:1,000), β-actin (Abcam, ab6276, 1:2,000), RPTOR (CST, 2280, 1:1,000), RICTOR (CST, 2114, 1:1,000), mLST8 (CST, 3274, 1:1,000), ubiquitin (Santa Cruz, sc-8017, 1:800), TSC1 (CST, 6935, 1:1,000), NPRL2 (CST, 37344, 1:1,000), histone H1 (Abcam, 11079, 1:1,000), EGFR (Santa Cruz, sc-373746, 1:800), AKT (CST, 9272, 1:1,000), p-AKT (CST, 4060, 1:1,000), mouse anti-goat IgG-HRP (Santa Cruz, sc-2354, 1:10,000), anti-rabbit IgG, HRP-linked antibody (CST, 7074, 1:10,000), mouse anti-rabbit secondary-HRP (Santa Cruz, sc-2357, 1:2,500) or recombinant anti-mouse (Santa Cruz, sc-516102, 1:2,500), anti-mouse IgG, HRP-linked antibody (CST, 7076, 1:10,000), normal rabbit IgG (CST, 2729, IP: 2 μg), normal mouse IgG (Santa Cruz, sc-2025, IP: 2 μg). siRNA transfection The indicated cells were transfected using Dharmafect 1 (Horizon) according to the manufacturer’s instructions. .. The following siRNAs were used: On-Target plus control siRNA (nontargeting, Dharmacon), On-Target plus pooled set of four siRNAs for CCNA2 (M-003205-020005), FZR1 (L-004086-00-0005), EMI1 (M-012434-01-0005), CCNF (custom, 5′-GCACCCGGUUUAUCAGUAAUUUU-3′), BTRC (encoding βTrCP) (L-003463-00-0005 and L-003490-00-0005), PFKFB3 (L-006763-00-0005), TSC1 (L-003028-00-0005), NPRL2 (L-01564500-0005), CDH1 3′ UTR (Qiagen, SI04955265|S1 (1027417) at final concentrations of 20 nM unless noted.
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