Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: 3H-glutamate uptake activity in human astrocytes. Human astrocyte cultures in 24-well plates (1×105 cells/well, 300 µl DMEM) were untreated or pretreated with DPI (300 nM) for 3h prior to IL-1β ± IFN-γ exposure for 24h followed by addition of 3H-glutamate (10 nM) for 10 min before harvesting for quantification. Data presented are mean ± MSE of triplicates from four separate experiments using astrocytes derived from different brain tissue specimens. nd: not done; *p<0.05 vs. untreated control, †p<0.05 vs. IL-1β or IL-1β+IFN-γ correspondingly.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Activity Assay, Derivative Assay

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: ROS production in human astrocytes. Human astrocyte cultures in 96-well plates (104 cells/well, 100 µl DMEM) were untreated (C) or treated with either A) IL-1β (0.01 to 10 ng/ml) ± IFN-γ (200 U/ml); B) IFN-γ (2 to 200 U/ml) ± IL-1β (10 ng/ml) for 24h;C) IL-1β (10 ng/ml) alone or in combination with IFN-γ (200 U/ml) for 3, 8 and 24h; or D) IFN-γ (200 U/ml), IL-1β (10 ng/ml) or IL-1β + IFN-γ for 24h. After washing, cultures were incubated with H2DCFDA (20 µM) for 45 min and read at Ex485nm and Em538nm (A–C) or microphotographed under fluorescent microscope (D). Data presented are mean ± MSE of triplicates of three separate experiments using astrocytes derived from different brain tissue specimens. *p<0.05 vs. respective (A) IL-1β, (B) IFN-γ alone or (C) untreated control.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Incubation, Microscopy, Derivative Assay

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: Inhibition of ROS production in human astrocytes. Human astrocyte cultures in 96-well plates (104 cells/well, 100 µl DMEM) were untreated (C) or pretreated with DPI (0.03 – 1 µM) overnight prior to IL-1β (10 ng/ml) + IFN-γ (200 U/ml) exposure for 24h before being washed and incubated with H2DCFDA (20 µM) for ROS measurement. Data presented are mean ± MSE of triplicates of three separate experiments using astrocytes derived from different brain tissue specimens. nd: not done; *p<0.05 vs. untreated control, tp<0.05 vs. IL-1β + IFN-γ.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Inhibition, Incubation, Derivative Assay

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: Mitochondrial ROS production in human astrocytes. Human astrocyte cultures in 4-well chamber slide (2×104 cells/well, 500 µl DMEM) were untreated (Control) or pretreated with DPI (1 µM) overnight prior to IL-1β (10 ng/ml) exposure for 24h before being stained with MitoSOX™ Red mitochondrial superoxide indicator. After washing, cells were counterstained with Hoechst 33342, washed and mounted in warm buffer to be microphotographed under a fluorescent microscope.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Staining, Microscopy

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: Effects of IL-1β and IFN-γ on antioxidant enzymes in human astrocytes. Total RNA (1 µg) or cell culture lysates collected from human astrocyte cultures in 12-well plates (106 cells/well, 600 µl DMEM) untreated or treated with IL-1β (10 ng/ml) ± IFN-γ (200 U/ml) for 24h were DNase treated and reverse transcribed to cDNA followed by qPCR or electrophoresed, transblotted and probed, respectively, to assess A) SOD2 and B) catalase expression. Densitometric measurement (normalized to β-actin) of the bands was shown. Data presented are mean ± MSE of three separate experiments using astrocytes derived from different brain tissue specimens. *p<0.05 vs. untreated control.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Cell Culture, Reverse Transcription, Expressing, Derivative Assay

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: IL-1β- and IFN-γ-induced signaling pathways in human astrocytes. Human astrocyte cultures in 12-well plates (106 cells/well, 600 µl DMEM) were untreated (as control) or treated with IL-1β (10 ng/ml) ± IFN-γ(200 U/ml) for 15, 30 and 60 min before collecting cell lysates to be electrophoresed, transblotted to nitrocellulose membrane and probed for p38 or p44/42 MAPK, Stat1 or β-actin (as internal control). Densitometric measurement (normalized to total p38 or p44/42 or β-actin) of the bands was shown. Data presented are representative of three to five separate experiments using astrocytes derived from different brain tissue specimens. All targets of the same experiment were electrophoresed and analyzed in individual blot without re-probing.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Membrane, Derivative Assay

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: Involvement of p38 MAPK in ROS production and glutamate uptake in human astrocytes. Human astrocyte cultures in 96-well plates (104 cells/well, 100 µl DMEM) were untreated (as control) or pretreated with A) SB203580 (p38 MAPK inhibitor) and SB202474 (as negative control) or B) U0126 (ERK1/2 MAPK inhibitor) and U0124 (as negative control) for 1h prior to IL-1β (10 ng/ml) + IFN-γ (200 U/ml) exposure for 24h before being washed and incubated with H2DCFDA (20 µM) for ROS measurement. C) Human astrocyte cultures in 24-well plates (1×105 cells/well, 300 µl DMEM) were untreated (as control) or pretreated with SB203580 (p38 MAPK inhibitor) and SB202474 (as negative control) for 1h prior to IL-1β (10 ng/ml) + IFN-γ (200 U/ml) exposure for 24h followed by addition of 3H-glutamate (10 nM) for 10 min before harvesting for quantification. Data presented are mean ± MSE of triplicates of three to five (A & B) and three (C) separate experiments using astrocytes derived from different brain tissue specimens. *p<0.05 vs. untreated control; †p<0.05 vs. IL-1β+IFN-γ.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Negative Control, Incubation, Derivative Assay

Journal: Neurochemical research

Article Title: Reactive Oxygen Species From Human Astrocytes Induced Functional Impairment and Oxidative Damage

doi: 10.1007/s11064-013-1123-z

Figure Lengend Snippet: ROS-induced 8-isoprostane production in human astrocytes. Human astrocyte cultures in 96-well plates (104 cells/well, 100 µl DMEM) were untreated (C) or pretreated with DPI (1 µM) overnight prior to IL-1β ± IFN-γ exposure for 24h before harvesting culture supernatants for 8-isoprostane measurement. Data presented are mean ± MSE of triplicates of three separate experiments using astrocytes derived from different brain tissue specimens. nd: not done; *p<0.05 vs. untreated control; †p<0.05 vs. IL-1β or IL-1β+IFN-γ correspondingly.

Article Snippet: Reagents The following reagents were purchased from the indicated sources: Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), Hanks’ balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), 3,3’-diaminobenzidine, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H–tetrazolium bromide (MTT), diphenyleneiodonium (DPI) (Sigma-Aldrich, St. Louis, MO); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); 3 H-glutamate (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ); acrylamide/bis-acrylamide gel and protein assay (Bio-Rad, Hercules, CA); gentamicin, Fungizone ® , RNase inhibitor, SuperScript™ III reverse transcriptase, Hoechst 33342, MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA); RNeasy mini kit (Qiagen, Valencia, CA); DNase (Ambion, Austin, TX); random hexmer and oligo (dT) 12–18 (Gene Link, Hawthorne, NY); SYBR ® Advantage ® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); CDP-Star substrate (Applied Biosystems, Foster City, CA); anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega, Madison, WI); human recombinant IL-1β and IFN-γ (R&D Systems, Minneapolis, MN); anti-p38, anti-extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK, anti-Stat1(Tyr701) and (Ser727) and anti-β-actin antibodies (Cell Signaling, Beverly, MA); anti-SOD2 and anti-catalase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); H 2 DCFDA, SB203580 (an inhibitor of p38 MAPK), SB202474 (negative control of SB203580), U0126 (an inhibitor of MAP kinase [MEK]1/2, upstream of ERK1/2), U0124 (negative control of U0126) and N G -monomethyl-L-arginine (N G MA, inhibitor of nitric oxide synthase) (EMD Biosciences, La Jolla, CA); 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI).

Techniques: Derivative Assay

Journal: Cell Reports

Article Title: HNF4A modulates glucocorticoid action in the liver

doi: 10.1016/j.celrep.2022.110697

Figure Lengend Snippet:

Article Snippet: 25μg of chromatin (using Nanodrop-measured concentration) was incubated overnight at 4°C with a GR antibody cocktail (ProteinTech 24050-1-AP (lot 00044414) and Cell Signaling D8H2 (lot 2) (2μl of each per IP reaction)).

Techniques: Recombinant, SYBR Green Assay, Sequencing, Software