Our recent study has described this method detailly [2 (link)]. The primary antibodies used in this present study included NOX2 (ab129068, 1 : 2000, Abcam, USA), AT1 (381666, 1 : 1000, Zenbio, Chengdu, China), ACE (24743-1-AP, 1 : 1000, proteintech, China), Bax (200958, 1 : 1000, Zenbio, Chengdu, China), MMP13 ((820098, 1 : 1000, Zenbio, Chengdu, China), C-caspase-3 (#9664, 1 : 1000, Cell Signaling Technology, Inc. USA), p53 (sc-393031, 1 : 500, Santa Cruz, Bio. Lnc, USA), Klotho (382164, 1 : 1000, Zenbio, Chengdu, China), Bcl2 (381702,, 1 : 1000, Zenbio, Chengdu, China), COX-2 (#12282, 1 : 1000, Cell Signaling Technology, Inc. USA), MMP-3 (380816, 1 : 1000, Zenbio, Chengdu, China), aggrecan (ab36861, 1 μg/mL, Abcam, USA), iNOS (#20609, 1 : 1000, Cell Signaling Technology, Inc. USA), Nrf2 (221102, 1 : 1000, Zenbio, Chengdu, China), HO-1 (#43966, 1 : 1000, Cell Signaling Technology, Inc. USA), type II collagen (collagen II (1 : 1000, ab34712, Abcam), SOD1 (#37385, 1 : 1000, Cell Signaling Technology, Inc. USA), NLRP3 (381207, 1 : 1000, Zenbio, Chengdu, China), ASC (340097, 1 : 1000, Zenbio, Chengdu, China), and GAPDH (5174, 1 : 1000, Cell Signaling Technology, Inc. USA). The secondary antibodies were purchased from Zenbio (380172, 511103, Zenbio, Chengdu, China).
Bcl2 associated x (bax)
Manufactured by ZenBio
Sourced in China
The Bax is a centrifuge designed for general laboratory applications. It features a compact and durable construction, allowing efficient separation of samples. The Bax operates at adjustable speeds to accommodate a variety of sample volumes and densities.
Automatically generated - may contain errors
Lab products found in correlation
Ab36861, by Abcam (2 mentions)
Erk1 2, by ZenBio (2 mentions)
P erk1 2, by ZenBio (2 mentions)
Caspase 3, by ZenBio (2 mentions)
P akt, by ZenBio (2 mentions)
Ab129068, by Abcam (1 mentions)
Aggrecan, by Abcam (1 mentions)
Mmp13, by ZenBio (1 mentions)
Sc 393031, by Santa Cruz Biotechnology (1 mentions)
Nlrp3, by ZenBio (1 mentions)
Ab34712, by Abcam (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
C caspase 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Phospho nf κb, by Cell Signaling Technology (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab283568, by Abcam (1 mentions)
10 protocols using bcl2 associated x (bax)
1
Comprehensive Antibody Panel for Oxidative Stress
2
Protein Extraction and Western Blotting of 4T1 Cells
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Total protein of murine 4T1 cells was extracted using RIPA lysis buffer (50 mM Tris, 150 mM sodium chloride, 1% NP-40, 0.25% deoxycholate) supplemented with protease and a phosphatase inhibitor cocktail (Beyotime Biotechnology). Equal amounts of protein were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA) by electroblotting. The membranes were probed with primary antibodies against B cell lymphoma 2-associated X (BAX; 1:1000, Zen-bioscience, Chengdu, Sichuan, China), IκBα, phospho-IκBα, NF-κB, phospho-NF-κB (1:1000 Cell Signaling Technology). Blots were developed with HRP-conjugated secondary antibodies and chemiluminescent substrates on Kodak X-ray film. The density of electrophoresis bands was quantified for statistical analysis.
3
Detailed Immunohistochemistry Antibody Protocol
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These two methods have been depicted in detail in our previous study [28 (link)]. The primer sequences are summarized in Table 1 . The primary antibodies in this study included CGRP (ab283568, 1 : 1000, Abcam, USA), CALCRL (861587, Zen Bio, Chengdu, China), RAMP1(385544, Zen Bio, Chengdu, China), Bax (200958, 1 : 1000, Zen Bio, Chengdu, China), Bcl2 (381702, 1 : 1000, Zen Bio, Chengdu, China), Cleaved-caspase3, iNOS (1 : 1000, Zen Bio, Chengdu, China), COX-2 (1 : 1000, Zen Bio, Chengdu, China), MMP3 (384995, 1 : 1000, Zen Bio, Chengdu, China), type I collagen (1 : 1000, ab34710, Abcam), type II collagen (1 : 1000, Zen Bio, Chengdu, China), aggrecan (ab36861, 1 μg/mL, Abcam, USA), NF-κB p65 (D14E12, #8242, Cell Signaling Technology, Inc., three Trask Lane Danvers, United States), p-p65 (Ser536; #3033, Cell Signaling Technology, Inc., three Trask Lane Danvers, United States), Ik-Ba (380682, 35 kDa; Zen Bio, Chengdu, China, 1 : 1,000), p-Ik-Ba (340776, 35 kDa; Zen Bio, Chengdu, China, 1 : 1,000), ERK1/2 (201245-4A4, 42/44 kDa; Zen Bio, Chengdu, China, 1 : 1,000), p-ERK1/2 (301245, 42/44 kDa; Zen Bio, Chengdu, China, 1 : 1,000), p38 (200782, 43 kDa; Zen Bio, Chengdu, China, 1 : 500), p-p38 (310069, 43 kDa; Zen Bio, Chengdu, China, 1 : 1,000), JNK (381100, 46/54 kDa; Zen Bio, Chengdu, China, 1 : 1,000), and p-JNK (380556, 46/54 kDa; Zen Bio, Chengdu, China, 1 : 1,000).
4
Liver Protein Extraction and Western Blot Analysis
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The refrigerated livers were washed with pre-cold PBS twice and centrifugation at 3000× g for 5 min at 4 °C, then removed the supernatant. Total protein extracts were obtained by homogenizing liver in RIPA lysis buffer (Sigma Aldrich) supplemented with protease inhibitor cocktail and phosphatase inhibitors. After centrifugation, the supernatant was collected and stored at − 80 °C. Protein concentration was determined by the BCA protein detection kit (Sangon Biotech, Shanghai, China). Western blot analysis was performed as previously described by Han et al. The primary antibodies were used: caspase-3 (ZenBio, Chengdu, China), caspase-9 (ZenBio), β-actin (Abcam, Cambridge, MA, USA), Bax (ZenBio), Bcl-2 (Santa Cruz, Heidelberg, Germany), LC3B (Sigma), P62 (Santa Cruz), beclin-1 (Sigma), PI3K/Akt/mTOR/70S6K protein and phosphorylated antibody were purchased from Bioss Biotechnology Co. Ltd. (Bioss, Beijing, China). The secondary antibodies used were as follows: mouse anti-rabbit (Sigma), goat anti-rabbit (Sigma), mouse anti-rabbit horseradish peroxidase (HRP) (Zenbio). The enhanced chemiluminescence (ECL) kit (Beyotime, Jiangsu, China) was used to capture the bands via a CanoScan LiDE 100 scanner (Canon, Tokyo, Japan), and western blots were analyzed by Image J software (Bethesda, MD, USA, 2007).
5
Quantitative Western Blotting Analysis
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For western blotting, whole‐cell protein samples were extracted and quantified then boiled at 95°C, 10 min. Then the samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred into a polyvinylidene fluoride (PVDF) membrane and incubated with primary antibodies overnight at 4°C, with specific primary antibodies against FAH (1:500, PA5‐42049, Invitrogen), Bax (1:1000, 380709, Zen Bioscience), Bcl2(1:1000, 15071T, Cell Signaling Technology) and GAPDH (TA802519, 1:2000, Origene) in Tris‐Buffered Saline Tween‐20 (TBST) containing 5% skim milk. After washed for three times with TBST, the membranes were incubated for 1 h at room temperature with a respective IgG‐HRP labeled second antibody (1:3000) in TBST. Antigens were revealed using a chemiluminescence assay (Pierce ECL Western Blotting Substrate, 32209, Thermo) and quantification of bands was achieved by densitometry using the Image J software.
6
Apoptosis and Autophagy Signaling Pathway
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The harvested cells were lysed by ultrasonication. The proteins were transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA) after gel electrophoresis (SDS-PAGE). The membranes were incubated at room temperature with 5% defatted milk powder for 1 h and then probed with the indicated primary antibodies: cleaved caspase-3, cleaved caspase-9, Bax, Bcl-2, LC3, beclin-1, P62, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38 (all from Zen Bioscience, Chengdu, China) at 4 ℃ overnight. Next, the following secondary antibody was employed and incubated 2 h at room temperature. The signal was developed by the ECL detection system, and the relative expression of proteins was analyzed using the Quantity One software.
7
Western Blot Analysis of Epithelial-Mesenchymal Transition and Apoptosis Markers
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Cells were harvested and lysed with RIPA and the protein concentration was detected by BCA protein assay (Solarbio,PC0020). Twenty mg total proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane, then incubated with the primary antibodies against E-cadherin (BA0475, 1:1500, BOSTER, China), N-cadherin (BA0673, 1:1000, BOSTER, China), Vimentin (PB9359, 1:1500, BOSTER, China), Bax (380,709, 1:2000, ZenBio, China), Bcl2 (381702,1:2000ZenBio, China), Cleaved-Caspase-3p17 (341,034, 1:1000ZenBio, China), Cleaved-Caspase-8-p18 (251,941, 1:2000ZenBio, China), β-catenin (R22820, 1:2000 ZenBio, China), c-Myc (R22809, 1:2000 ZenBio, China), Caspase-3 (R23315, 1:2000 ZenBio, China), MAL2 (BS-7175r, 1:1500, BIOSS BIOSS, China), GAPDH (GB11002, 1:2000, Servicebio, China) overnight. The membranes were subsequently incubated with the appropriate HRP-conjugated secondary antibodies (purchased from proteintech, SA00001-2, 1:10000) for 1.5 h, and signals were visualized using an ECL detection system.
8
Hippocampus Protein Expression Analysis
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Following behavioral testing, mice from each group were randomly chosen to be decapitated under deep anesthesia, and the bilateral hippocampal tissues were quickly dissected on the ice board. Protein was extracted with RIPA buffer (Solar bio, Beijing, China) following the manufacturer's instructions. The tissues were subjected to centrifugation at 4°C, 12 000 rpm for 10 min. Samples were separated using 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (PVDF) (Millipore Corporation, United States). Then, 5% skim milk in 0.1% Tween 20 in TBS (TBST) was used to block nonspecific binding for 1 h at room temperature. The blots were incubated overnight at 4°C with antibodies against Nrf2 (1:1000 Proteintech), HO‐1 (1:1000 Proteintech), NOX2 (1:1000 Proteintech), Bax (1:1000 ZEN BIO), Bcl‐2(1:1000 ZEN BIO), Caspase‐3 (1:1000 ZEN BIO), β‐actin (1:3000 ZEN BIO) as the loading control. On the next day, the membranes were washed with TBST (10 min × 3) and incubated with the secondary antibody (goat anti‐rabbit IgG, 1:3000, ZEN BIO) for 1 h. After washing the membranes, the relative densities of the protein bands were analyzed using an Odyssey imaging system (Li‐Cor Biosciences, Lincoln, NE, USA) and quantified using ImageJ analysis software (National Institutes of Health, Bethesda, MD).
9
Apoptosis Signaling Pathway Assay
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The reagents and antibodies that were used in our study included: bufotalin (HY-N0878, MCE, USA); Actin (AC026, Abclonal, China), Bcl2 (381702, Zenbio, China), BAX (R380709, Zenbio, China), BAD(R23582, Zenbio, China), caspase-3(A2156, Abclonal, China), cleaved caspase-3 (A19654, Abclonal, China), AKT (382804, Zenbio, China), p-AKT(310021, Zenbio, China), HRP Goat Anti-Rabbit IgG (AS014, Abclonal, China), HRP Goat Anti-Mouse IgG (AS003, Abclonal, China) and Cy3 Goat Anti-Rabbit IgG(AS007, Abclonal, China).
10
Western Blot Analysis of Protein Expression
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To extract cellular proteins, a lysis buffer (Thermo Fisher Scientific) was employed. A BCA protein assay kit (Beyotime) was used to determine the protein concentration. Subsequently, equal amounts of the protein were separated by SDS-PAGE, and then transferred onto polyvinylidene difluoride membranes. These membranes were blocked using 5% skimmed milk and incubated overnight at 4 ℃ with primary antibodies targeting specific proteins, including ICAM2 (Cell Signaling Technology), GAPDH (Proteintech), BAX (ZENBIO), BAD (ZENBIO), BCL2 (Proteintech), E-cadherin (Proteintech), N-cadherin (ZENBIO), Claudin 1 (Proteintech), Snail1 (Proteintech), B-catenin (Abmart), HA (Proteintech), and RDX (ZENBIO). After washing, the membranes were incubated with secondary antibodies (Proteintech) for 1 h at room temperature. They were then visualized using an enhanced chemiluminescence reagent (Meilunbio) and captured using a ChemiDoc Touch imaging system (Bio-Rad).
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