Mouse anti ser473akt

Equal amounts of total protein extracts were separated by SDS-PAGE and electrotransferred to nitrocellulose membrane (GE-Healthcare Europe, Milan, Italy). Membranes were probed with rabbit anti-NLRP3 (Abcam, Cambridge, UK), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Ser⁴⁷³Akt (Cell Signaling Technology, Danver, MA, USA), rabbit anti-total Akt (Cell Signaling Technology, Danver, MA, USA), rabbit anti-Ser⁹ GSK-3β (Abcam, Cambridge, UK), anti-total GSK-3β (Cell Signaling Technology, Danver, MA, USA), anti-Ser⁶⁶⁰ PKC and total PKC (Santa Cruz Biotechnology, Dallas, TX, USA), SOD2 (Novus Biologicals, Centennial, CO, USA), and NRF2 (Thermo Fisher Scientific, Whaltam, MA, USA) followed by incubation with appropriate HRP-conjugated secondary antibodies (BioRad). Proteins were detected with Clarity Western ECL substrate (BioRad, California, USA) and quantified by densitometry using analytic software (Quantity-One, BIO-RAD Image Lab Software.6.0.1.). Results were normalized with respect to densitometric value of mouse anti-tubulin (Abcam, Cambridge, UK), and autoradiograms showing statistically significant differences in terms of gel-loading homogeneity were excluded from the following biomarkers analyses.

Total proteins extracts were separated by SDS-PAGE and blotted to nitrocellulose membrane (GE-Healthcare Europe, Milano, Italy). Membranes were incubated with rabbit anti-NLRP3 (Abcam, Cambridge, UK), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-GSDMDC1 (Santa Cruz Biotechnology), rabbit anti-IL-1β (Santa Cruz Biotechnology), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Tyr²⁰⁴ ERK1/2 (Cell Signaling Technology), rabbit anti-total ERK1/2 (Cell Signaling Technology), mouse anti-Ser⁴⁷³ Akt (Cell Signaling Technology), rabbit anti-total Akt (Cell Signaling Technology), rabbit anti-Ser⁹ GSK-3β (Abcam, Cambridge, UK), anti-total GSK-3β (Cell Signaling Technology), rabbit anti-mitochondrial transcription factor A (mtTFA) (Novus Biologicals, Cambridge, UK), mouse anti-nuclear respiratory factor-1 (NRF-1) (Santa Cruz Biotechnology), and mouse anti-sarcomeric mitochondrial creatine kinase (sMtCK) (Santa Cruz Biotechnology) and then probed with proper HRP-conjugated secondary antibodies (BioRad). Clarity Western ECL substrate (BioRad) was used for protein detection and quantification was performed by densitometric analysis (Quantity-One, Bio-Rad software). Data were normalized according to the related antitubulin densitometric values.