Bax Protein

We performed Western blots to detect Mcl-1, Bcl-xl, Bcl-2, and Bax in the total protein extracts of Daoy, D283, and D341 cells. GAPDH was used as the loading control. Total cell proteins were extracted by lysing in a buffer containing 50 mM Tris-HCl (pH 8.0), NP-40 (1.0%), NaCl (150 mM), and 0.5% of the Roche protease inhibitor cocktail. This was followed by 8 min of centrifugation (10,000× g), and the quantification of total cell protein lysates by the Bradford protein assay, as previously reported [58 (link)]. We used denaturing SDS-PAGE to run protein samples, and transferred the samples to PVDF (Polyvinylidene Difluoride) membranes. These membranes were then blocked with 3% milk-Tween 20, and incubated overnight with primary antibody [Cell Signalling antibodies: Bax (2774S), Bid (2002S), Mcl-1 (4572S), Bcl-xl (2762S); ThermoFisher Scientific: Bcl-2 (MA5-11757); and Santa Cruz Biotechnology: GAPDH (sc-47724)] at 4 °C. After three washes, membranes were incubated with HRP-conjugated secondary antibody [Sigma-Aldrich: Anti-Rabbit IgG (A6154), and Jackson ImmunoResearch: Peroxidase-conjugated affinpure Goat anti-Mouse IgG, Light chain specific (115-035-174)] (1 h, room temperature). The enhanced chemiluminescence (ECL) kit from Amersham-Pharmacia Biotech was used for blot development, as previously reported [59 (link),60 (link)].

Liver tissue sections were heated at 60°C for 25 min in an oven (Venticell, MMM, Einrichtungen. Germany) and then deparaffinized in xylene and rehydrated using graded alcohol. The process of antigen retrieval was performed in 10 mM sodium citrate buffer boiled in a microwave. Immunohistochemistry staining steps were performed following the manufacturer’s instructions (DakoCytomation, USA). In brief, endogenous peroxidase was blocked using 0.03% hydrogen peroxide sodium azide for 5 min. Tissue sections were washed gently with wash buffer and then incubated with Bcl-2–associated X protein (Bax) (1:500), Proliferating Cell Nuclear Antigen (PCNA) (1:200) and anti-apoptotic protein Bcl2 (1:50) biotinylated primary antibodies for 15 min. Sections were gently washed with wash buffer and kept in the buffer bath in a humid chamber. A sufficient amount of streptavidin-HRP was then added and incubated for 15 min followed by washing. Diaminobenzidine-substrate chromagen was added to the sections and incubated for over 7 min followed by washing and counterstaining with hematoxylin for 5 sec. The sections were then dipped in weak ammonia (0.037 M/L) 10 times, washed and cover slipped. Positive antigens stained brown under light microscopy.

Cells (approximately 5 × 10⁶) were lysed in 200 µL RIPA Lysis and Extraction Buffer (Thermo Fisher; #89900), and the tubes were kept on ice for 20 min. The resulting cell lysates were centrifuged at 13,000g for 20 min, and the total protein levels in the supernatants were quantified using a Nanodrop instrument. Equal amounts of protein were separated by electrophoresis on 10% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher; #88520) and blocked with 5% fat-free milk for 1 h. The membranes were then probed with primary antibodies, including anti-Bcl2 (Abcam, Shanghai, China; #ab241548), anti-Bcl2A1 (Thermo Fisher; #TA806463), anti-Bcl-xL (Abcam; #ab32370), anti-Bcl-w (Thermo Fisher; #MA515076), anti-Mcl1 (Abcam; #ab246684), anti-p65 (Abcam; #ab32536), anti-p50 (Abcam; #ab283688), anti-p300 (Abcam; #ab10485), anti-NCOA3 (Abcam; #ab133611), anti-Flag (Abcam; #ab49763), anti-Myc (Abcam; #ab32), anti-ERα (Sigma-Aldrich; #SAB4500813), anti-ERβ (Abcam; #ab3576), anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Abcam; #ab8245), anti-Bax (Bcl-2 associated X-protein) (Abcam; #ab270742), anti-Bak (Bcl-2 homologous antagonist/killer; Abcam; #ab32371), and anti-caspase 9 (Cell Signaling, Shanghai, China; #9502). After washing 5 times with PBS containing Tween20 (PBST), the membranes were probed with secondary antibodies, including anti-mouse IgG H&L (Abcam; #ab205719) and anti-Rabbit IgG H&L (Abcam; #ab205718). Protein bands were detected using the Pierce™ ECL Western Blotting Substrate Kit (Thermo Fisher; #32109). Protein band intensity was normalized using ImageJ software (Fiji version 1.44a).

The same surgical procedures were performed as described above and rats were assigned into 2 groups at 10 min after CPR (Fig. 1C): (1) The post-resuscitation normoxic therapy group (n = 6) included those that were successfully resuscitated from 10-min asphyxia CA and treated with inhaled 30% oxygen (CA-Normo) following the initial 10 min of 100% oxygen, and (2) The post-resuscitation hyperoxia group (n = 6) included those treated with inhaled 100% oxygen (CA-Hypero) during the entire observational period. For all rats, the brain and heart tissues were collected at 2 h after CPR for mRNA extraction, followed by complementary DNA (cDNA) synthesis and real-time PCR. Additionally, tissues of control (naive) rats were collected to create a reference for mRNA gene expression.
RNA isolation, reverse transcription, and real-time PCR analysis for the brain, heart, and lung samples extracted at 2 h after CA were performed according to manufacturer instructions. Briefly, total RNAs were extracted and reverse transcribed using TRIzol Reagent® (Invitrogen, Carlsbad, CA) and SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (Invitrogen, Carlsbad, CA), respectively. Real-time PCR was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems™, Waltham, MA) on the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany). All primers were purchased from Thermofisher: Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, TaqMan Assay ID: Rn01775763_g1), Interleukin-1 beta (Il1b, Rn00580432_m1), Interleukin-6 (Il6, Rn01410330_m1), Transforming growth factor-beta 1 (Tgfb1, Rn00572010_m1), Intercellular adhesion molecule-1 (Icam1, Rn00564227_m1), Nuclear factor-kappa beta 1 (Nfkb1, Rn01399583_m1), Tumor necrosis factor (Tnf) receptor-associated factor-6 (Traf6, Rn01512911_m1), Caspase-3 (Casp3, Rn00563902_m1), Caspase-9 (Casp9, Rn00581212_m1), Epidermal growth factor (Egf, Rn00563336_m1), and B-cell leukemia/lymphoma-2 (Bcl2) associated X protein (Bax, Rn02532082_g1).