Immunoblotting of Inflammatory Proteins

SH-SY5Y cells and VM organotypic cultures were treated with a lysis buffer and protein concentrations were estimated by the Bio-Rad protein assay using bovine serum albumin as standard, as previously described [10 (link)]. Specific primary antibody anti-iNOS (BD Transduction 610329; 1/500), anti-COX2 (Cell Signaling #4842 1/500), anti-Bid (1/500; Santa Cruz Biotechnology), anti-Bad (1/500; Santa Cruz Biotechnology), anti-IκBα (Santa Cruz Biotechnology sc-371; 1/500), anti-NFκB p65 (Santa Cruz Biotechnology sc-372; 1/500), anti-Bcl2 (Santa Cruz Biotechnology sc-7382; 1/500) and anti-Bax (Santa Cruz Biotechnology sc-7480; 1/500) were mixed in 1× PBS, 5% w/v nonfat dried milk, 0.1% Tween-20 (PMT) and incubated at 4°C, overnight. Signals were detected as described above [11 (link)].

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Paterniti I., Campolo M., Siracusa R., Cordaro M., Di Paola R., Calabrese V., Navarra M., Cuzzocrea S, & Esposito E. (2017). Liver X receptors activation, through TO901317 binding, reduces neuroinflammation in Parkinson’s disease. PLoS ONE, 12(4), e0174470.

Publication 2017

protein assay anti-iNOS anti-COX2 anti-Bid anti-Bad anti-IκBα anti-NFκB p65 anti-Bcl2 anti-Bax

Antibody Assay Bcl2 Bovine serum albumin Buffer Cells Cox2 Inos Iκbα Milk Nfκb p65 Organotypic cultures Protein Rad protein Sc 372 Tween 20

Corresponding Organization : Saint Louis University

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Variable analysis

independent variables

Treatment with a lysis buffer

dependent variables

Protein concentrations estimated by the Bio-Rad protein assay
Signals detected for the following proteins: iNOS, COX2, Bid, Bad, IκBα, NFκB p65, Bcl2, Bax

control variables

Bovine serum albumin as standard for the Bio-Rad protein assay
Primary antibody dilutions (1/500) for iNOS, COX2, Bid, Bad, IκBα, NFκB p65, Bcl2, Bax

controls

Positive control: Not specified
Negative control: Not specified

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