Recombinant human TNF-α, hemin and ATP were purchased from Sigma Aldrich. NIH3T3 cells were treated with reagents diluted in complete medium at concentrations determined by preliminary experiments and detailed below. Anti-hE5NT (4G4, Novus Biologicals), anti-hHO-1 (EP1391Y, Epitomics) and anti-hENTPD1 (BU61, Santa Cruz) primary antibodies, Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit (Life Technologies) secondary antibodies were used for immunofluorescence analysis. anti-hHO-1 (EP1391Y, Epitomics), anti-hE5NT (EPR6115, LifeSpan BioSciences), anti-hENTPD1 (HPA014067, Sigma Aldrich), anti-β-actin (AC-15, Sigma Aldrich)), anti-Nf-kB1 p105/p50 (D4P4D, Cell Signaling) and anti-Lamin B2 (EPR9701(B), Abcam) primary antibodies were used for immunoblotting analysis. Phycoerythrin (PE)-conjugated anti-hE5NT (BD Biosciences) and Alexa Fluor 647-conjugated anti-hENTPD1 (Life Technologies) antibodies were used for FACS analysis and cell sorting.
Cinti A., De Giorgi M., Chisci E., Arena C., Galimberti G., Farina L., Bugarin C., Rivolta I., Gaipa G., Smolenski R.T., Cerrito M.G., Lavitrano M, & Giovannoni R. (2015). Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro. PLoS ONE, 10(10), e0141933.
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