The commercially available mitochondrial division inhibitor, Mdivi-1, was from Sigma-Aldrich (Saint-Louis, MO, USA) and utilized for treating SK-HEP-1 and Huh7 cells, with DMSO (Sigma-Aldrich) as the control group. Translation inhibitor cycloheximide chase was also from Sigma-Aldrich for assessing protein stability. The primary antibodies against cleaved (c)-caspase-3 (ab2302, Abcam), total (t)-caspase-3 (ab13847, Abcam), c-caspase-6 (ab2326, Abcam), t-caspase-6 (ab185645, Abcam), Bax (ab32503, Abcam), Bcl-2 (ab32124, Abcam), E-cadherin (ab40772, Abcam), N-cadherin (ab76057, Abcam), MMP2 (ab37150, Abcam), MMP7 (ab5706, Abcam), DRP1 (ab184247, Abcam), PDI1 (ab4644, Abcam), CDK1 (ab18, Abcam), H-RAS (G12V) (ab140571, Abcam), LZTR1 (ab106655, Abcam), c-Myb (ab109127, Abcam), and GAPDH (ab245356, Abcam), as well as secondary antibodies were all obtained from Abcam (Cambridge, MA, USA). Antibodies against p-DRP1 (S616) (#3455S, Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2 (#4370S, Cell Signaling Technology), and ERK1/2 (#4695S, Cell Signaling Technology) were obtained from Cell Signaling Technology (Danvers, MA, USA).
Ab245356
Manufactured by Abcam
Sourced in United States
Ab245356 is a laboratory equipment product manufactured by Abcam. It serves as a tool for conducting various scientific experiments and analyses. The core function of this product is to provide a reliable and efficient solution for researchers and scientists in their laboratory work. Further details on the intended use or specific applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (4 mentions)
Sds page, by Bio-Rad (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Ab37150, by Abcam (2 mentions)
Ab40772, by Abcam (2 mentions)
Ab5706, by Abcam (2 mentions)
Ab76057, by Abcam (2 mentions)
Ab13847, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ab32572, by Abcam (2 mentions)
Ab32539, by Abcam (2 mentions)
Ab2324, by Abcam (2 mentions)
Ab184247, by Abcam (1 mentions)
Ab2326, by Abcam (1 mentions)
Ab185645, by Abcam (1 mentions)
Ab140571, by Abcam (1 mentions)
Ab109127, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
9 protocols using ab245356
1
Mdivi-1 Inhibits Mitochondrial Division
2
Wnt/β-catenin Signaling Pathway Activation
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Transfected DU145 or LNCaP cells with or without LiCl treatment were lysed in RIPA buffer (Thermo Fisher Scientific) with additional blends of protease inhibitors (Roche, Mannheim, Germany). Lysate was collected and subjected to SDS-PAGE (Bio-Rad, Hercules, CA, USA), after which were transferred onto PVDF membranes (Bio-Rad). Upon incubation with Blocking One reagent (Nacalai Tesque, Kyoto, Japan), membranes were blotted by primary antibodies against CTNNB1 (SAB2701829, Sigma-Aldrich), β-catenin (ab32572, Abcam), CCND1 (SAB1405510, Sigma-Aldrich), CDK2 (ab32147, Abcam), c-MYC (ab32072, Abcam), and GAPDH (ab245356, Abcam) and then by HRP-labeled secondary antibodies. The signals were monitored after washing using the ECL Western blot kit (Thermo Fisher Scientific). Assay was undertaken in at least triplicate.
3
Western Blot Analysis of Signaling Proteins
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TPC-1 or BCPAP cells were lysed using RIPA protein extraction regent (Beyotime, Shanghai, China). Protein concentration was evaluated through a BCA protein assay kit (Abcam, Cambridge, USA). Proteins were later separated using 10% SDS-PAGE (Bio-Rad), followed by being shifted to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were probed by use of primary antibodies against LPAR3 (1: 2000 dilution, PA5-27074, Invitrogen), EGFR (1: 2000 dilution, ab52894, Abcam), p-PI3K (1: 1000 dilution, ab182651, Abcam), PI3K (1: 1000 dilution, ab191606, Abcam), p-AKT (1: 1000 dilution, ab131443, Abcam), AKT (1: 10,000 dilution, ab179463, Abcam), p-mTOR (1: 2000 dilution, ab109268, Abcam), mTOR (1: 2000 dilution, ab2732, Abcam) and GAPDH (1: 10,000 dilution, ab245356, Abcam) at 4 °C overnight. After being washed utilizing PBS supplemented with 0.1% Tween-20 (Sigma-Aldrich), membranes were cultivated with HRP-tagged secondary antibodies (1: 10,000 dilution, ab205718, Abcam). Protein bands were assayed by an ECL Substrate Western Blot Detection system (Pierce, Rockford, IL, USA) with the Molecular Imager ChemiDoc XRS system (Bio-Rad).
4
Western Blot Analysis of Apoptosis and MMPs
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Western blot was implemented on the basis of prior description [17 (link)]. Primary antibodies against Bax (ab32503), Bcl-2 (ab32124), Cleaved caspase-3 (ab2302), Total caspase-3 (ab13847), Cleaved caspase-9 (ab2324), Total caspase-9 (ab32539), MMP2 (ab37150), MMP7 (ab5706), MMP9 (ab38898), LARP4 (ab241489) and GAPDH (ab245356) applied were purchased from Abcam (Cambridge, USA).
5
Western Blot Analysis of DNA Damage Markers
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Cells were lysed through RIPA buffer (Invitrogen). After centrifugation, the collected proteins were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinylidene fluoride membranes (PVDF; Bio-Rad). The membranes were blocked with 5% nonfat milk and consecutively probed with primary antibodies including anti-γH2AX (ab2893, Abcam, Cambridge, USA), anti-cleaved PARP (ab32064, Abcam), anti-CHEK2 (ab109413, Abcam), anti-FOXM1 (ab180710, Abcam) and anti-GAPDH (ab245356, Abcam) as an internal control, followed by treated with secondary antibody. Finally, immunoreactivity was assessed via enhanced chemiluminescence (ECL; Bio-Rad).
6
Western Blot Analysis of EMT Markers
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7
Quantification of BCL2L2 protein expression
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Protein was detached in RIPA buffer (Applygen) and quantitated by a protein quantitative kit (Beyotime). 20 μg/lane Protein was segregated by sodium dodecyl sulfate-polyacrylamide gel and then transferred on polyvinylidene fluoride membrane (Solarbio). The membrane was blockaded in 5% non-fat milk, and then nurtured with the antibody against BCL2L2 (ab190952, 1:2000, Abcam, Cambridge, MA, USA) overnight and a secondary antibody IgG labeled by horseradish peroxidase (HRP) (ab97200, 1:20,000, Abcam) for 2 h. GAPDH (ab245356, 1:5000, Abcam) was used as a loading control. After hatching with ECL reagent (Amyjet Scientific, Wuhan, China), the blots were visualized by ChemiDoc XRS+ system (Bio-rad Laboratories, Richmond, California, USA) and analyzed Image J v1.8 software (NIH, Bethesda, MD, USA).
8
Evaluating Apoptosis Markers Expression
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Equal amounts of protein were applied to 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and subsequently transferred to PVDF membranes (Bio-Rad). Membranes were blocked by 5% non-fat dry milk in Tris Buffered Saline with Tween® 20 (TBS-T; Sigma-Aldrich) and then incubated at 4 °C with primary antibodies overnight to reveal the expression of them. Next, membranes were washed with TBS-T, followed by incubated for 1 h with specific secondary antibodies. Peroxidase activity were visualized with the enhanced chemiluminescence substrate system (ECL; Santa Cruz Biotechnology, Santa Cruz, CA, United States) and the bands were quantified using Quantity One (Bio-Rad). Primary antibodies against cleaved-caspase3 (ab32042, Abcam, Cambridge, USA), total caspase3 (ab32351, Abcam), cleaved-caspase9 (ab2324, Abcam), total caspase9 (ab32539, Abcam), Bax (ab32503, Abcam) β-catenin (ab32572, Abcam) and GAPDH (ab245356, Abcam) were employed, separately.
9
Western Blot Analysis of Myocardial Proteins
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Total protein from myocardial tissues or cells was extracted using Protein Lysis Buffer (Beyotime, Shanghai, China). The protein concentration was determined using BCA Protein Assay Kit (Beyotime). Equal amounts of protein were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with defatted milk and incubated with primary antibodies overnight at 4°C. After washing with PBS, the membranes were incubated with secondary antibodies goat antirabbit IgG (1 : 10,000, ab6721) for 2 h at room temperature. Finally, the protein bands were visualized with enhanced chemiluminescence detection kits (Amersham Pharmacia Biotech, UK) and analyzed by ImageJ software. The primary antibodies against Bax (1 : 1000, ab32503), collagen I (1 : 1000, ab34710), SH2B3 (1 : 1000, ab191904), collagen III (1 : 1000, ab7778), Bcl-2 (1 : 1000, ab182858), fibronectin (1 : 1000, ab2413), and GAPDH (ab245356) were purchased from Abcam (Cambridge, USA) and GAPDH acted as a loading control. This assay was performed for 3 times (n = 3).
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