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2 protocols using hybond p polyvinylidene difluoride pvdf blotting membranes

1

Western Blot Analysis of Inflammatory Signaling

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Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), L-glutamine penicillin/streptomycin, and anti-α-tubulin monoclonal antibodies (mAbs) were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). LPS (Escherichia coli 0127:B8), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Lamin B1 and anti-iNOS polycloncal antibody (pAb) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-TNF-α, anti-JNK, anti-phospho-c-JNK (Thr183/Tyr185), anti-phospho-p44/p42 ERK (Thr202/Tyr204), anti-phospho-p38 MAPK (Thr180/Tyr182) pAbs, and anti-phospho-p65 (Ser536), anti-p65, anti-IκBα, anti-ERK and anti-p38 MAPK mAbs were purchased from Cell Signaling (Danvers, MA, USA). Anti-IL-1β pAb was purchased from BioVision (Milpitas, CA, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG were purchased from Amersham (Buckinghamshire, UK). The Western blotting detection reagent of enhanced chemiluminescence (ECL) and Hybond™-P polyvinylidene difluoride (PVDF) blotting membranes were purchased from GE Healthcare Life Sciences (Waukesha, WI, USA).
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2

Cellular Response to Inflammatory Stimuli

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Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), L-glutamine/penicillin/streptomycin, and anti-α-tubulin monoclonal antibodies (mAbs) were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). LPS and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma-Aldrich (St. Louis, MO, USA). Anti-iNOS, COX2, and Nrf2 polyclonal antibodies (pAbs) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). An anti-HO-1 pAb was purchased from Enzo (Farmingdale, New York, NY, USA). The anti-phospho-p65 (Ser536) mAb was purchased from Cell Signaling (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG) and sheep anti-mouse IgG were purchased from Amersham (Buckinghamshire, U.K.). Enhanced chemiluminescence (ECL) Western blotting detection reagent and Hybond-P polyvinylidene difluoride (PVDF) blotting membranes were purchased from GE Healthcare Life Sciences (Waukesha, WI, USA).
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