The soluble protein extracts were separated by SDS-PAGE on a 10%–20% gradient gel and transferred to a Polyvinylidene difluoride (PVDF) membrane. Each blot membrane was incubated as per instruction with primary antibodies. The primary antibodies against Bcl2 (ab692), Bax (ab7977), Caspase 3 (ab32351), Caspase 9 (ab32539), and β-actin (ab63982) were purchased from Abcam. The blots were incubated with secondary goat anti-rabbit antibody (Abcam, ab63982) for 1 h at room temperature. Protein bands were detected on X-ray film using an enhanced chemiluminescence detection system.
Ab692
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab692 is a lab equipment product designed for general laboratory use. It serves as a tool for researchers and scientists in their experimental work. The core function of Ab692 is to facilitate the collection, processing, and analysis of samples as required by various laboratory protocols and procedures. Further details on the specific intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (20 mentions)
Ab2302, by Abcam (8 mentions)
Ab13847, by Abcam (7 mentions)
Ab6721, by Abcam (6 mentions)
Ab8245, by Abcam (5 mentions)
Ab32539, by Abcam (4 mentions)
Ab181602, by Abcam (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ab8226, by Abcam (4 mentions)
Ab7977, by Abcam (3 mentions)
Ab182651, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab6276, by Abcam (3 mentions)
Ab92552, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Ab38449, by Abcam (3 mentions)
Ab16667, by Abcam (3 mentions)
Ab134175, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab32351, by Abcam (2 mentions)
50 protocols using ab692
1
Western Blot Analysis of Apoptosis Markers
2
Exosome and Cell Protein Analysis
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Both exosomes and cells were lysed using the radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shifted onto a polyvinylidene fluoride membrane, and blocked with 5% non-milk. Next, the membranes were incubated with primary antibodies against CD9 (1:5000, ab68418, Abcam, Cambridge, MA, USA), CD63 (1:2000, ab68418, Abcam), Matrix metallopeptidase 9 (MMP-9) (1:1000, ab38898, Abcam), Vimentin (1:5000, ab92547, Abcam), B-cell lymphoma-2 (Bcl-2) (1:1000, ab692, Abcam), BCL2-associated X protein (Bax) (1:1000, ab32503, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GADPH) (1:10,000, ab181602, Abcam), followed by interaction with secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). Finally, signals were visualized with the chemiluminescence chromogenic substrate (Beyotime).
3
Western Blot Analysis of Apoptosis and Metabolism Regulators
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Cells were lysed by lysis buffer (10 mM Tris, 1 m MEDTA,1% Triton X-100, 1 mM Na3VO4, 20 μg/mL aprotinin, 20 μg/mL leupeptin, 1 mM dithiothreitol, and 50 μg/mL phenylmethylsulfonyl fluoride). Cell lysates were subjected to 10% SDS-PAGE and proteins were transferred to the PVDF membrane by an electro-transfer unit. Membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies included anti-Bcl-2 (ab692, Abcam; 1:2000), anti-caspase-3 (ab32351; 1:2000), anti-Bax (#89477, cell signaling technology, Beverly, MA, USA; 1:2000), anti-PGC1-α (ab54481; 1:2000), anti-NRF1 (ab34682; 1:2000), anti-PKC (ab181558; 1:2000), anti-HMGB1 (ab18256; 1:2000), anti-RAGE (ab216329; 1:2000), and anti-NFκB (ab16502; 1:2000). Following incubation of primary antibodies, the membranes were rinsed three times and incubated with corresponding secondary antibodies. The immunoreactive bands were developed using a Western-Ready™ ECL Substrate Plus Kit (426316, BioLegend, San Diego, CA, USA) and detected by MultiGel-21® image system. β-actin was used as an internal control.
4
Western Blot Analysis of Brain Proteins
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The total protein was extracted with radio-Immunoprecipitation Assay (RIPA) lysate from the brain tissue or A172 cells of each group. The BCA method was used to determine the concentration of the proteins, and 30 µg of the protein samples was separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride (PVDF) membranes using the wet-transfer method. The membranes were blocked with 5% skim milk powder and incubated with primary antibody (1:1,000) at 4 °C overnight. The primary antibody was then washed away, the membranes were invubated with the goat anti-rabbit secondary antibody (Abcam, 1:5,000) at room temperature for 1h. After electrochemical luminescence (ECL) visualization, the relative expression levels of the target bands were derived by the gray scale ratio with the internal reference. The primary antibodies included anti-glial fibrillary acidic protein (GFAP) (Abcam, ab4674), anti-GAPDH (Abcam, ab76523), anti-Bcl-2 (Abcam, ab692), and anti-LC3B (Abcam, ab192890).
5
Western Blot Analysis of Apoptosis Markers
6
Western Blot Analysis of Bcl-2 Pathway
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Transfected cells were lysed by RIPA lysis buffer (Beyotime). Then the lysate was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membranes. Subsequently, the membranes were incubated with primary antibody against B-cell lymphoma-2 (Bcl-2) (1:1000, ab692, Abcam, Cambridge, MA, USA), BCL2-associated X protein (Bax) (1:1000, ab32503, Abcam), phosphorylated (p)-phosphoinositide 3-kinase (p-PI3K) (1:1000, ab182651, Abcam), PI3K (1:1000, ab40776, Abcam), p-protein kinase B (p-AKT) (1:1,000, 9271, Cell Signaling Technology, Boston, MA, USA), AKT (1:1,000, 9272, Cell Signaling Technology), as well as β-Actin (1:1,000, 4967, Cell Signaling Technology). After interaction with secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). Experiments were performed three times, and immunoreactive bands were visualized using electrochemiluminescence.
7
Evaluating Cholesterol and Statin Metabolism Biomarkers
8
Protein Expression Analysis in Melanoma Cells
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The total protein was extracted from cultured melanoma cells using the RIPA buffer (Beyotime, Shanghai, China) to detect the expression level of CCT3. The protein concentration of each sample was determined using the BCA protein assay kit (Blue Skies, Shanghai, China). 10% SDS-PAGE gel electrophoresis was used for separating total proteins, then the proteins were transferred onto polyvinylidene difluoride membranes. After the membranes were blocked with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies: mouse anti-GAPDH (1:2000, Santa Cruz, sc-2005), mouse anti-β-actin (1:10000, Abcam,ab6276), rabbit anti-CDK1 (1:300, Abcam, ab32094), rabbit anti-FOXM1 (1:400, Abcam, ab180710), rabbit anti-MCM-2 (1:1000, Abcam, ab109271), rabbit anti-NFKBIA (1:300, Abcam, ab7217), rabbit anti-PIM1 (1:100, Abcam, ab75776), and rabbit anti-SKP2 (1:300, Abcam, ab183039), rabbit anti-Bcl-2 (1:500, Abcam, ab692), rabbit anti-Bax (1:500, Abcam, ab32503), and rabbit anti-Cleaved PARP (1:1000, Abcam, ab32064). The membranes were then washed with TBST 3 times and incubated with the secondary antibody (Yeasen, Shanghai, China) for 2 h at room temperature. Bands were visualized using a chemiluminescent HRP substrate (Millipore, MA, USA) and an electrogenerated chemiluminescence imaging system (Tanon, Shanghai, China) 26 .
9
Evaluating Protein Expression in Prostate Cancer Cells
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Protein expression in DU145 cells treated with either CK (control) or si-NK, astaxanthin or si-STAT3, or astaxanthin and si-STAT3 was evaluated using Western blotting. DU145 cells were seeded in 6-well plates and cultured to 80% confluency. The cells were then treated with CK (control) or si-NK, astaxanthin or si-STAT3, or astaxanthin and si-STAT3 for 24 h, followed by protein extraction. An equivalent of 30 μg protein was subjected to SDS-PAGE and Western blotting. The primary antibodies were diluted as follows: Bcl-2(26 kDa) 1:1000 (ab692, Abcam, Cambrige, MA, USA), Bax (21 kDa) 1:1000 (ab7977, Abcam, Cambrige, MA, USA), Caspase3 (32 kDa) 1:1000 (ab32351, Abcam, Cambrige, MA, USA), Caspase9 (46 kDa) 1:1000 (ab32539, Abcam, Cambrige, MA, USA), NF-κB p65 (65 kDa) 1:1000 (ab76302, Abcam, Cambrige, MA, USA), JAK2 (131 kDa) 1:1000 (ab92552, Abcam, Cambrige, MA, USA) and Stat3 (88 kDa) 1:1000 (ab119352, Abcam, Cambrige, MA, USA). Each Western blotting assay was repeated at least two times.
10
Western Blot Analysis of Hedgehog Pathway Proteins
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Protein was detected using the bicinchoninic acid kit (cat. no. 20201ES76; Yeason Biotechnology Co. Ltd.); samples were diluted in deionized water to achieve a loading quantity of 30 µg protein per lane. Proteins were separated via SDS-PAGE (12% gel) transferred onto a nitrocellulose membrane, which was sealed using 5% skim milk powder at 4°C and kept overnight. Diluted primary anti-rabbit polyclonal antibodies were added to the cells, and included Shh (1:1,000, ab53281, Abcam), Gli1 (1:1,500, ab151796, Abcam), Ptch1 (1:1,500, ab53715, Abcam), basic fibroblastic growth factor (b-FGF; 1:200, ab99979, Abcam), transforming growth factor β (TGF-β; ab31013, Abcam), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; 1:2,000, ab32503, Abcam), Bcl-2 (1:500, ab692, Abcam), and GAPDH (1:2,500, ab9485, Abcam). The rabbit anti-human monoclonal secondary antibody (1:200, bs-0361R-HRP, BIOSS Company, Beijing, China) was used then added. Subsequently, protein bands were visualized using electrochemical luminescence (ECL808-25, Biomiga, Inc., San Diego, CA, USA) and analyzed by Image J software (version 2.1.4.7; National Institutes of Health Inc.).
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