Bcl2 associated x (bax)

FFPE tissue slides were double stained for BRCA1, BRCA2, p15, Bax and Tec (Abcam Inc., Cambridge MA) with Ubiquitin (Millipore, Temecula, CA). A second set of slides were stained for BRCA1, BRCA2, p15, Bax and Tec (Enzo Life Sciences, Farmingdale, NY) with Ubiquitin (Millipore, Temecula, CA). BRCA1, BRCA2, p15, Bax and Tec were detected using donkey anti rabbit Alexa Fluor 488 (Jackson Immuno Research Laboratories Inc. West Grove, PA). Ubiquitin was detected using donkey anti mouse Alexa Fluor 594 (Jackson Labs. West Grove, PA). All slides were stained with the nuclear stain DAPI (Molecular Probes, Eugene, OR). The fluorescence intensity of stained protein of interest was measured quantitatively using a 40× objective and a standard exposure time of 800ms using a Nikon 400 fluorescent microscope with three filters (FITC-green, Texas Red, and Tri-Color), and the Nikon morphometric system. The results were displayed as a graph attached to the immunofluorescent photography using a screen snip.

RIPA reagent (Solarbio) and BCA kit (#23235, Thermo Fisher Scientific) were chosen to conduct protein extraction and concentration quantification, several times. The different proteins of every sample were separated using gel electrophoresis and then transferred onto polyvinylidene difluoride (PVDF, Bedford, MA, USA) membranes. Then, they were covered and maintained in 5% fat-free milk at room temperature for 2 h. The primary antibodies included Bax (No. ADI-AAM-140-E, Enzo Life Sciences, Inc., Farmingdale, NY, USA), Bcl-2 (No. ADI-AAM-072-E), TNF-α (No. LS-B6067, LifeSpan BioSciences, Seattle, WA, USA), IL-6 (No. LS-C746886), IL-1β (No. 503513, San Diego, CA, USA), p-IκBα (phospho Ser32/Ser36, No. GTX79042, GeneTex, Irvine, CA, USA), t-IκBα (No. GTX27545), p-p65 (phospho Ser468, No. GTX32256), t-p65 (No. GTX107678), and β-actin (No. GTX629630). After incubation with above primary antibodies at 4℃ overnight, secondary antibodies were used to block at room temperature for 1 h. Signals of protein were developed with an ECL™ Western Blotting Analysis system (Sigma). The intensity was analyzed by ImageJ software (version 146; National Institutes of Health, Bethesda, MD, USA).