Rats were sacrificed 4 weeks after burn injuries by an overdose of Zoletil 50. Lumbar 3,4,5 (L345) spinal cords were collected, and the dorsal horn area and skin were separated, frozen in liquid nitrogen, and stored at -80°C. L345 dorsal horn specimens and skin were homogenized in an ice-cold lysis buffer, T-PER Tissue Protein Extraction Reagent (Thermo Scientific) with one tablet of Complete Protease Inhibitor Cocktail (Roche) per 25 mL and then centrifuged (15,000 g) for 30 minutes at 4°C. Each protein concentration in the supernatant was measured using bovine serum albumin as the standard. The procedures and analyses of Western blots were performed through the same method as our previous report [26 (link)]. The primary antibodies of COX-2 (1:1000, Cell Signaling, Danvers, MA), iNOS (1:1000, Abcam, Cambridge, MA), and nNOS (1:1000, Abcam, Cambridge, MA), protein kinase B (AKT) (1:1000, Cell Signaling, Boston, MA), p-AKT (1:1000, Cell Signaling, Boston, MA), Bcl-2 Associated X protein (Bax) (1:1000, Proteintech Group, Chicago, IL), Bcl-2 (1:1000, Abcam, Cambridge, MA), and β- actin (1:20000 dilution, Sigma-Aldrich, Saint Louis, MO) were used in this study.
Bcl 2 associated x protein bax
Manufactured by Proteintech
Sourced in United States, United Kingdom, China
Bcl-2-associated X protein (Bax) is a protein that plays a key role in the regulation of apoptosis, a process of programmed cell death. Bax is a member of the Bcl-2 protein family and acts as a pro-apoptotic factor, promoting the initiation of apoptosis under certain cellular conditions.
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Lab products found in correlation
Bcl 2, by Abcam (2 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (2 mentions)
Bcl 2, by Proteintech (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Protein kinase b akt, by Cell Signaling Technology (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Tunel kit, by Beyotime (1 mentions)
Protease inhibitor, by Roche (1 mentions)
β actin, by Boster Bio (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Bcl 2, by Bioworld Technology (1 mentions)
C myc, by Proteintech (1 mentions)
Enhanced chemiluminescence, by Wuhan Boster Biological Technology (1 mentions)
Bca assay, by Beyotime (1 mentions)
Sirt1, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
8 protocols using bcl 2 associated x protein bax
1
Molecular Mechanisms in Burn-Induced Pain
2
Carmofur-Induced Apoptosis Mechanism
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Carmofur (Car) (Cat# S1289) was purchased from Selleck (Houston, TX, USA). Doxepin (DOX) and Vitamin C (Vc) were obtained from XinYi Pharmaceutical Co. Ltd. (Shanghai, China) and LiJun Pharmaceutical Co. Ltd. (Xi’an, Shaanxi, China), respectively. Rabbit polyclonal antibody against ACDase (Cat# 11274-1-AP,) and Bcl-2 Associated X Protein (bax) (Cat# 50599-2-Ig) were purchased from Proteintech Group (Rosemont, IL, USA). Rabbit monoclonal antibody specific for B cell lymphoma/lewkmia-2 (bcl-2) (Cat# ab182858) and mouse monoclonal antibody against ASM (Cat# ab74281) were purchased from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against Cer (Cat# ALX-804-196) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The TUNEL kit (Cat# C1088) was purchased from Beyotime (Shanghai, China). Malondialdehyde (MDA) testing kit (Cat# A003-1) and superoxide dismutase (SOD) activity testing kit (Cat# A001-1) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The SP Histostain-Plus Kits (Cat# SP-9001, SP-9002, ZLI-9018) were purchased from ZSGB-Bio (Beijing, China).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using radio immunoprecipitation assay buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitors (Roche Diagnostics, Basel, Switzerland) at 4°C for 30 min, and western blot analysis was performed as described previously (12 (link)). Primary antibodies against B-cell lymphoma 2 (Bcl-2; cat. no. 12789-1-AP; 1:2,000; Protein Tech Group, Inc., Chicago, IL, USA), Bcl-2-associated X protein (Bax; cat. no. BM3964; 1:500; Boster Biological Technology, Pleasanton, CA, USA) and β-actin (cat. no. 4967S; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) were incubated with the membranes overnight at 4°C. Following the primary incubation, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG secondary antibodies (Sigma-Aldrich; Merck KGaA).
4
Protein Expression Analysis in Cells
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The cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) at 4°C on ice for 30 min. After centrifugation in 13,400 × g for 5 min at 4°C, 30 µg of protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The blots were incubated with rabbit anti-human cyclin D2 (1:400; cat. no. 10934-1; Proteintech Group, Inc.)/Bcl-2-associated X protein (Bax; 1:400; cat. no. BS2583; Bioworld Technology, Inc.)/Bcl-2 (1:400; cat. no. 12789-1; Proteintech Group, Inc.)/c-Myc (1:400; cat. no. BS2462; Bioworld Technology, Inc.)/β-actin (1:400; cat. no. BS-0061R; Beijing Biosynthesis Biotechnology Co., Ltd.) antibodies at 4°C overnight. Membranes were washed with TBS and Tween-20 (TBST) three times. HRP-labeled goat anti-rabbit IgG (1:6,000; cat. no. BS13278; Bioworld Technology, Inc.) was then added for 1 h at room temperature. Finally, signals were captured using enhanced chemiluminescence (Wuhan Boster Biological Technology, Ltd.) after three washes with 1X TBST. The assays were performed in triplicate. Densitometry was performed using a Gel Image System 4.2 (Tanon Science & Technology Co., Ltd.).
5
Western Blot Analysis of Synaptic Proteins
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Brain tissue or primary neuronal cells were homogenized in ice-cold lysis buffer with protease inhibitors and phosphatase inhibitors, and the protein concentrations were determined by the BCA assay (Beyotime Biotechnology, CHN). The samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, equal amounts (20–30 μg) of proteins were loaded per lane, transferred to 0.45-μm polyvinylidene difluoride (PVDF) membrane, and blocked for 2 h at room temperature. Afterward, the membranes were incubated overnight at 4°C with primary antibodies: postsynaptic density protein 95 (PSD-95) (1:1000, CST, United States), synapsin-1 (SYN-1) (1:1200, CST, USA), NeuN (1:1000, CST, United States), SIRT1 (1:1000, Abcam, UK), Nrf2 (1:1000, Abcam, UK), Bcl-2-associated X protein (Bax) (1:4000, Proteintech, United States), B-cell lymphoma 2 (Bcl-2) (1:800, Proteintech, United States), and β-actin (1:400, Santa Cruz, United States). Then, the membranes were incubated with secondary antibodies (1:10,000, Jackson ImmunoResearch, United States). Protein bands were visualized with enhanced chemiluminescence (ECL). Then, the intensity of the bands was quantified by ImageJ software (version 1.8.8, National Institutes of Health, United States) and corrected with the corresponding β-actin level. The results were expressed as values normalized to the loading sham.
6
Western Blot Analysis of Apoptosis-Related Proteins
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Caco-2 cells were collected with RIPA (Beyotime, China) with phenylmethylsulfonyl fluoride (PMSF). Identical amounts of protein were divided by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% milk and incubated with primary antibodies for B-cell lymphoma 2 (Bcl-2, Proteintech, China), Bcl-2-associated X protein (Bax, Proteintech, China), cyclin D1 (Proteintech, China), SIRT1 (Proteintech, China), caspase-3 (Cell Signaling, US), p-p65 (Bioworld Technology, USA), p65 (Bioworld Technology, USA), p-IκB (Bioworld Technology, USA), IκB (Bioworld Technology, USA), and GAPDH (Sigma, USA) at 4 °C overnight. Then, the membranes were exposed to the corresponding secondary antibodies for 1 h. Finally, the proteins were visualized using a Bio-Rad imaging system.
7
Protein Extraction and Western Blot Analysis
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For total protein extraction, the contusion-side cortex of animals was lysed completely in ice-cold radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China) that contained protease inhibitor with centrifugation at 12000 g at 4°C for 20 minutes. The supernatant was collected, and then protein concentration was estimated using a bicinchoninic acid assay kit (23227; Thermo Fisher Scientific). After denaturing at 95°C for 5 minutes in 5× loading sodium dodecyl sulfate (SDS) buffer (Invitrogen), a total of 30 µg protein of each sample was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Blocking buffer containing 5% non-fat milk was used to block the membranes for 1 hour at room temperature. Then the membranes were incubated with primary antibodies against β-actin (1 : 10000), NMNAT2 (Abcam, Cambridge, UK; 1 : 1000), BCL-2-associated X protein (Bax; 1 : 500; Protein Tech, Rosemont, IL, USA) at 4°C overnight. The next day, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase. Finally, the immunoblots were detected by enhanced chemiluminescence reagents (Millipore) in an imaging system (BioRad, Hercules, CA, USA). The relative protein intensity was analyzed using Image-J software (National Institutes of Health, Bethesda, MD, USA).
8
Western Blot Analysis of Cardiac Protein Expression
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Cardiac tissues and cellular proteins were lysed using radioimmunoprecipitation assay lysis buffer and quantified by bicinchoninic acid. After collecting the lysate supernatant, equal amounts of proteins were boiled for 5 min with loading buffer. Protein samples (30 µg per lane), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were transferred to polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA) and blocked with buffer (5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) buffer) for 1 h. The membranes were incubated with primary antibodies against STEAP4 (1:500 dilution; Novus Biological, CO, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000 dilution; Proteintech, Wuhan, China), B-cell lymphoma 2 (Bcl-2; 1:500 dilution; Proteintech), and Bcl-2-associated X protein (Bax; 1:500 dilution; Proteintech). Following washing 3 times (15 min each time) with TBST, the membranes were incubated with the corresponding secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit) for 1 h at 18-25°C. After washing 3 times, protein bands were displayed by using enhanced chemiluminescence reagent and quantified by Gel-Pro analyzer version 4.0 software (Media Cybernetics). GAPDH was used as a standard control.
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