Structured Review

Santa Cruz Biotechnology leupeptin
Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with <t>leupeptin</t> (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P
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1) Product Images from "Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells"

Article Title: Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells

Journal: Nature immunology

doi: 10.1038/s41590-017-0002-1

Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P
Figure Legend Snippet: Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Confocal Microscopy, Expressing, shRNA

Related Articles

Transfection:

Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

Article Title: Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7
Article Snippet: .. A 20 nM concentration of CXCL12-stimulated HEK293 cells or CXCR7 stably transfected HEK293 cells or IM-9 cells (2 × 107 ) were lysed in 20 mM triethanolamine (pH 8.0), 300 mM NaCl, 2 mM EDTA, 20% glycerol, 2% digitonin, 10 mM sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin for 30 min at 4°C and then centrifuged (15,000 × g , 15 min, 4°C) and processed for Western blot with anti-phospho-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA). .. Untransfected or CXCR7-stably transfected HEK293 cells (2.5 × 106 cells/ml) were resuspended in RPMI medium 1640 containing 10% FCS and 10 mM Hepes and incubated with Fluo-3 (Calbiochem, La Jolla, CA).

Stable Transfection:

Article Title: Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7
Article Snippet: .. A 20 nM concentration of CXCL12-stimulated HEK293 cells or CXCR7 stably transfected HEK293 cells or IM-9 cells (2 × 107 ) were lysed in 20 mM triethanolamine (pH 8.0), 300 mM NaCl, 2 mM EDTA, 20% glycerol, 2% digitonin, 10 mM sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin for 30 min at 4°C and then centrifuged (15,000 × g , 15 min, 4°C) and processed for Western blot with anti-phospho-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA). .. Untransfected or CXCR7-stably transfected HEK293 cells (2.5 × 106 cells/ml) were resuspended in RPMI medium 1640 containing 10% FCS and 10 mM Hepes and incubated with Fluo-3 (Calbiochem, La Jolla, CA).

Protease Inhibitor:

Article Title: ROCK Inhibition Activates MCF-7 Cells
Article Snippet: .. Immunoblotting The cells were lysed with a lysis buffer (Cell Signaling Technology, Beverly, MA) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3 VO4 , 1 µg/ml leupeptin, 1 mM PMSF, and a complete protease inhibitor cocktail tablet (Santa Cruz Biotechnology, Santa Cruz, CA). .. After high-speed centrifugation of the cell lysates, the protein concentration of the supernatants was determined using the Bradford assay (Bio-Rad, Hercules, CA).

Article Title: The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells
Article Snippet: .. The cells were then washed 3 times with ice-cold PBS and lysed for 30 min with RIPA lysis buffer [50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 100 mM phenylmethylsulfonyl fluoride, 1 μg/ml of leupeptin, 1 mM Na3VO4, and 1× Complete Protease Inhibitor Cocktail (Santa Cruz Biotechnology)]. .. Equal amounts of protein were loaded onto 10–15% SDS-PAGE gels, electrophoresed, and transferred onto PVDF membranes (Millipore, Bedford, MA).

Article Title: Prostate specific membrane antigen associates with anaphase promoting complex and induces chromosomal instability
Article Snippet: .. 100 μg of total protein from synchronized and mitotic block-released PC3 and PC3-PSMA cells lysed in 95 mM NaCl, 25 mM Tris, pH 7.4, 0.5 mM EDTA, 2% SDS, 1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml each of antipain, leupeptin, and pepstatin (protease inhibitor cocktail) were used for immunoblot analysis using anti-cyclin B1 (Santa Cruz Biotechnology Inc, Santa Cruz, CA). .. Proteins were detected by enhanced chemiluminescence (ECL) and quantified by densitometry.

Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

Flow Cytometry:

Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

Blocking Assay:

Article Title: Prostate specific membrane antigen associates with anaphase promoting complex and induces chromosomal instability
Article Snippet: .. 100 μg of total protein from synchronized and mitotic block-released PC3 and PC3-PSMA cells lysed in 95 mM NaCl, 25 mM Tris, pH 7.4, 0.5 mM EDTA, 2% SDS, 1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml each of antipain, leupeptin, and pepstatin (protease inhibitor cocktail) were used for immunoblot analysis using anti-cyclin B1 (Santa Cruz Biotechnology Inc, Santa Cruz, CA). .. Proteins were detected by enhanced chemiluminescence (ECL) and quantified by densitometry.

SYBR Green Assay:

Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

Concentration Assay:

Article Title: Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7
Article Snippet: .. A 20 nM concentration of CXCL12-stimulated HEK293 cells or CXCR7 stably transfected HEK293 cells or IM-9 cells (2 × 107 ) were lysed in 20 mM triethanolamine (pH 8.0), 300 mM NaCl, 2 mM EDTA, 20% glycerol, 2% digitonin, 10 mM sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin for 30 min at 4°C and then centrifuged (15,000 × g , 15 min, 4°C) and processed for Western blot with anti-phospho-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA). .. Untransfected or CXCR7-stably transfected HEK293 cells (2.5 × 106 cells/ml) were resuspended in RPMI medium 1640 containing 10% FCS and 10 mM Hepes and incubated with Fluo-3 (Calbiochem, La Jolla, CA).

Incubation:

Article Title: Mechanisms of G2 Arrest in Response to Overexpression of p53
Article Snippet: .. Cell lysates prepared with 50 mM HEPES, pH 7.0, 250 mM NaCl, 0.1% nonidet P-40, 10% glycerol, 1 mM phenylmethanesulfonyl fluoride, 2 μg/ml aprotinin, 25 μg/ml leupeptin, 5 μg/ml pepstatin A, and 1 mM DTT, containing equal quantities of protein, were incubated with either a CDC2-specific monoclonal antibody (17; Santa Cruz Biotechnology), a polyclonal antibody raised against the C terminus of CDC2 (Babco, Richmond, CA) , or a monoclonal antibody specific for cyclin B1 (GNS1; Santa Cruz Biotechnology). .. Immune complexes were isolated with protein A– conjugated sepharose (Pharmacia, Bridgewater, NJ).

Labeling:

Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

Gel Extraction:

Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

Western Blot:

Article Title: Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7
Article Snippet: .. A 20 nM concentration of CXCL12-stimulated HEK293 cells or CXCR7 stably transfected HEK293 cells or IM-9 cells (2 × 107 ) were lysed in 20 mM triethanolamine (pH 8.0), 300 mM NaCl, 2 mM EDTA, 20% glycerol, 2% digitonin, 10 mM sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin for 30 min at 4°C and then centrifuged (15,000 × g , 15 min, 4°C) and processed for Western blot with anti-phospho-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA). .. Untransfected or CXCR7-stably transfected HEK293 cells (2.5 × 106 cells/ml) were resuspended in RPMI medium 1640 containing 10% FCS and 10 mM Hepes and incubated with Fluo-3 (Calbiochem, La Jolla, CA).

Lysis:

Article Title: ROCK Inhibition Activates MCF-7 Cells
Article Snippet: .. Immunoblotting The cells were lysed with a lysis buffer (Cell Signaling Technology, Beverly, MA) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3 VO4 , 1 µg/ml leupeptin, 1 mM PMSF, and a complete protease inhibitor cocktail tablet (Santa Cruz Biotechnology, Santa Cruz, CA). .. After high-speed centrifugation of the cell lysates, the protein concentration of the supernatants was determined using the Bradford assay (Bio-Rad, Hercules, CA).

Article Title: The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells
Article Snippet: .. The cells were then washed 3 times with ice-cold PBS and lysed for 30 min with RIPA lysis buffer [50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 100 mM phenylmethylsulfonyl fluoride, 1 μg/ml of leupeptin, 1 mM Na3VO4, and 1× Complete Protease Inhibitor Cocktail (Santa Cruz Biotechnology)]. .. Equal amounts of protein were loaded onto 10–15% SDS-PAGE gels, electrophoresed, and transferred onto PVDF membranes (Millipore, Bedford, MA).

Immunofluorescence:

Article Title: Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells
Article Snippet: .. For immunofluorescence staining, HEK293T cells in which Atad3a was knocked down or not were seeded in the culture chamber and were treated with or without 10 μM leupeptin for 3 h, then fixed with 4% PFA, permeabilized with 0.1% Triton and stained with anti-Tom20 (sc-17764, Santa Cruz) and anti-Lamp2a (ab18528, Abcam). ..

Staining:

Article Title: Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells
Article Snippet: .. For immunofluorescence staining, HEK293T cells in which Atad3a was knocked down or not were seeded in the culture chamber and were treated with or without 10 μM leupeptin for 3 h, then fixed with 4% PFA, permeabilized with 0.1% Triton and stained with anti-Tom20 (sc-17764, Santa Cruz) and anti-Lamp2a (ab18528, Abcam). ..

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    Santa Cruz Biotechnology leupeptin
    Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with <t>leupeptin</t> (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P
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    Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

    Journal: Nature immunology

    Article Title: Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells

    doi: 10.1038/s41590-017-0002-1

    Figure Lengend Snippet: Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

    Article Snippet: For immunofluorescence staining, HEK293T cells in which Atad3a was knocked down or not were seeded in the culture chamber and were treated with or without 10 μM leupeptin for 3 h, then fixed with 4% PFA, permeabilized with 0.1% Triton and stained with anti-Tom20 (sc-17764, Santa Cruz) and anti-Lamp2a (ab18528, Abcam).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Confocal Microscopy, Expressing, shRNA

    Role of Calpain in ApoE-Induced ABCA1 Protein ( A ) Macrophages were treated with 100 μg/ml cycloheximide (CHX) or vehicle medium in the presence or absence of 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h incubation with 10 μg/ml apoE3 or medium alone. ( B ) Macrophages were treated with vehicle medium (Veh), 50 μM ALLN, 1 mM leupeptin (Lpeptn), 10 μM aprotinin (Aprotn), 20μM pepstatin (Peptn), or 5 μM lactacystin (Lactyn) for 2 h. ( C ) Macrophages were treated with 50 μM ALLN or vehicle medium in the presence or absence of 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h incubation with 10 μg/ml apoE3 or control medium. ( D ) Macrophages were treated with or without 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h treatment with 10 μg/ml apoE3 or control medium alone. The cells were then treated with 3 μg/ml of calpain-1 or vehicle medium. ABCA1 (A1) protein was detected by immunoblotting, and quantified relative to GAPDH ( A–C ) or CD-MPR ( D ). ( E ) Macrophages were treated with 4 μM Akt X or vehicle medium for 2 h, followed by 2 h treatment with 10 μg/ml apoE3 or control medium. Calpain activity was measured with a calpain activity kit. Data represent mean ± SE of 3–4 independent experiments. ( A ) * P

    Journal: Biochemical and biophysical research communications

    Article Title: Akt Isoform-Dependent Regulation of ATP-Binding Cassette A1 Expression by Apolipoprotein E

    doi: 10.1016/j.bbrc.2016.06.031

    Figure Lengend Snippet: Role of Calpain in ApoE-Induced ABCA1 Protein ( A ) Macrophages were treated with 100 μg/ml cycloheximide (CHX) or vehicle medium in the presence or absence of 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h incubation with 10 μg/ml apoE3 or medium alone. ( B ) Macrophages were treated with vehicle medium (Veh), 50 μM ALLN, 1 mM leupeptin (Lpeptn), 10 μM aprotinin (Aprotn), 20μM pepstatin (Peptn), or 5 μM lactacystin (Lactyn) for 2 h. ( C ) Macrophages were treated with 50 μM ALLN or vehicle medium in the presence or absence of 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h incubation with 10 μg/ml apoE3 or control medium. ( D ) Macrophages were treated with or without 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h treatment with 10 μg/ml apoE3 or control medium alone. The cells were then treated with 3 μg/ml of calpain-1 or vehicle medium. ABCA1 (A1) protein was detected by immunoblotting, and quantified relative to GAPDH ( A–C ) or CD-MPR ( D ). ( E ) Macrophages were treated with 4 μM Akt X or vehicle medium for 2 h, followed by 2 h treatment with 10 μg/ml apoE3 or control medium. Calpain activity was measured with a calpain activity kit. Data represent mean ± SE of 3–4 independent experiments. ( A ) * P

    Article Snippet: Pepstatin A (sc-45036), lactacystin (sc-3575), ALLN (sc-221136), batimastat (sc-203833), leupeptin-HCl (sc-215242), aprotinin (sc-3595), Akt inhibitor X (sc-203811), Akt inhibitor XI (sc-221229), PI3K inhibitor , and antibodies against ABCA1 (sc-58219), β-actin (sc-47778), GAPDH (sc-32233), CD-MPR (sc-34573), p-Akt (sc-7985-R), Akt (sc-8312), and Sp1 (sc-14027) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Incubation, Activity Assay