Western Blot Analysis of Cellular Fractions

Extracts of the cytosol and nucleus were prepared, as previously mentioned [84 (link),87 (link),88 (link),89 (link),90 (link)]. The following primary antibodies were used: anti-Bax (1:500, Santa Cruz Biotechnology, #sc7480), anti-Bcl-2 (1:500, Santa Cruz Biotechnology, #sc7382), anti-Iκbα (1:500, Santa Cruz Biotechnology, #sc-1643), and anti-nfκb (1:500, Santa Cruz Biotechnology, #sc8414) in 1× PBS, 5% w/v non-fat dried milk, and 0.1% Tween 20, at 4 °C overnight [91 (link),92 (link),93 (link),94 (link)]. For the cytosolic fraction, Western blots were also probed with antibody against β-actin protein (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). The same methods were used for nuclear fraction with lamin A/C (1:500, Sigma-Aldrich Corp., Milan, Italy) [95 (link),96 (link)]. Signals were examined with an enhanced chemiluminescence (ECL) detection system reagent, according to the manufacturer’s instructions (Thermo, Monza, Italy). The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTM XRS+ software [84 (link),91 (link),97 (link),98 (link),99 (link)].

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Genovese T., Siracusa R., Fusco R., D’Amico R., Impellizzeri D., Peritore A.F., Crupi R., Gugliandolo E., Morabito R., Cuzzocrea S., Trovato Salinaro A., Cordaro M, & Di Paola R. (2021). Atrazine Inhalation Causes Neuroinflammation, Apoptosis and Accelerating Brain Aging. International Journal of Molecular Sciences, 22(15), 7938.

Publication 2021

anti-Bax anti-Bcl-2 anti-Iκbα anti-nfκb antibody against β-actin protein lamin A/C BIORAD ChemiDocTM XRS+ software

Actin Antibodies Antibody Bcl 2 Chemiluminescence Cytosolic Densitometry Iκbα Lamin a c Milk Nfκb Nucleus Protein Protein 1 Tween 20 Western blots

Corresponding Organization : University of Catania

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of Bax protein
Levels of Bcl-2 protein
Levels of IκBα protein
Levels of NF-κB protein

control variables

Methods used to prepare cytosol and nuclear extracts as described in references [84], [87], [88], [89], and [90]
Primary antibodies used at 1:500 dilution in 1x PBS, 5% w/v non-fat dried milk, and 0.1% Tween 20, incubated at 4°C overnight as described in references [91], [92], [93], and [94]
β-actin protein as a loading control for cytosolic fraction
Lamin A/C as a loading control for nuclear fraction

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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