*4.7. Automated Western Immunoblotting*

Before blotting, the protein was quantified using the bicinchoninic acid (BCA) method. Simple western immunoblotting was performed on a simple wes system (ProteinSimple, San Jose, CA, USA) using a Size Separation Master Kit with Split Buffer (12–230 kDa) according to the manufacturer's standard instruction and using specific antibody. Anti-PTEN (phospho S380) antibody [EP2138Y] (ab76431), Anti-PTEN antibody [Y184] (ab32199), Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283), Anti-AKT1/2/3 antibody [EPR16798] (ab179463), Anti-IκBα (phospho S32+S36) antibody (ab12135), Anti-IκBα antibody (ab32518), Anti-NF-κB-p65 antibody (ab32536), Anti-iNOS antibody (ab178945), Anti-COX2 antibody (ab179800) were purchased from Abcam(Cambridge, USA), Anti-p27kip1 antibody (2552), Anti-Lamin A/C antibody (2032S,) and anti-β-actin (4970S) antibody were obtained from CST (Danvers, MA, USA). Compass software (version 4.0.0, ProteinSimple, San Jose, CA, USA) was used to program the Peggy Sue and for presentation (and quantification) of the western immunoblots. Output data were displayed from the software calculated average of seven exposures (5–480 s).
