Perihematomal brain tissues in ICH groups and corresponding area in sham group were collected for western blot anslysis. We extracted proteins of mitochondria, according to the manufacturer’s protocol from Isolation Kit for Tissue protocol (Pierce Biotechnology, Rockford, IL, United States). The detailed procedures of western was reported in one of our previous study.31 The primary antibodies were listed as follows: DJ-1 (1:15000, Abcam ab76008), p-Akt (1:2000, CST 4060s), Akt (1:3000, CST 4691), p-IKK (1:1000, CST 2697), IKK (1:1000, CST 2682), NF-κB p65 (1:3000, ab86299), Bax (1:1000, Abcam ab32503), Bcl-2 (1:500, Abcam ab59348), caspase-3 (1:500, Abcam ab13847), cleaved caspase-9 (1:1500, Abcam ab25758) and β-actin (1:5000, Abcam ab8226), NDUFS8 (1:2000, Abcam ab180183), COX IV (1:1000, CST 4844).
Ab25758
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab25758 is a primary antibody designed for use in immunochemical applications. It is a highly specific and sensitive reagent targeted towards a specific antigen. The product details, including the target antigen and recommended applications, are available on the Abcam website.
Automatically generated - may contain errors
Lab products found in correlation
Ab13847, by Abcam (3 mentions)
Ab2324, by Abcam (3 mentions)
Ab59348, by Abcam (2 mentions)
Ab115777, by Abcam (2 mentions)
Ab32042, by Abcam (2 mentions)
Ab202068, by Abcam (2 mentions)
Ab7315, by Abcam (2 mentions)
Ab4051, by Abcam (2 mentions)
Ab227503, by Abcam (2 mentions)
Ab31370, by Abcam (2 mentions)
A0216, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
A0208, by Beyotime (2 mentions)
Ab32503, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
Ab22609, by Abcam (1 mentions)
Ab76128, by Abcam (1 mentions)
Ab171095, by Abcam (1 mentions)
9 protocols using ab25758
1
Mitochondrial Protein Analysis in ICH
2
Western Blotting of Brain Proteins
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Lysis of cells was performed using lysis buffer (200 μL/well). Constituents of lysis buffer were NaCl (150 mM), Tris (pH 7.6, 50 mM), Triton X-100 (1%), including phosphatase and protease inhibitors. Extracted proteins (40 μg of the total proteins) were separated by performing SDS–PAGE (7.5%). Procedure of western blotting was carried out as described by Waraich et al. and Run et al. [26 (link),27 (link)]. Following antibodies were used for western blotting: anti-GFAP, Abcam (ab7260); anti-Oligo2, Abcam (ab136253); anti-Iba1, Abcam (ab5076); anti-beta actin, Abcam (ab115777); anti-caspase-3, Abcam (ab13847); anti-cleave caspase-3, Abcam (ab32042); anti-caspase-9, Abcam, (ab202068); anti-cleave caspase-9, Abcam (ab25758); anti-tau, Abcam (ab76128); anti-tau phospho, Abcam (ab109390); anti-CaMKII, Abcam (ab22609); and anti-CaMKII phospho, Abcam (ab171095). Samples of protein were transferred to nitrocellulose membrane post-electrophoresis. Membrane was blocked by bovine serum albumin or skim milk for 2 h and later incubated overnight with primary antibody (Ab) at 4°C. After washing, membrane was incubated with secondary-Ab (anti-rabbit/mouse Ig-G conjugated to horseradish peroxidase) for 1 h at room temperature. Enhanced chemiluminescence was carried out to visualize protein expression. All western blot experiments were in triplicate. ImageJ was used for protein quantification.
3
Investigating Apoptosis Pathways in Cells
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Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) was purchased form Gibco (Life technologies, Shanghai, China). An antibody that recognizes both the long and short isoforms of cFLIP (cFLIPL and cFLIPS) (sc-5276) was purchased from santa cruz (Santa Cruz, USA). Another one against caspase-8 was from Alexis Biochemicals. Other antibodies against caspase-3 (ab90437), caspase-9 (ab25758), PCNA (ab2426) and fibronectin (ab2413) were from abcam (Cambridge, UK). Emodin, streptozotocin (STZ), D-glucose, mannitol, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk), bortezomib and other chemicals were purchased from Sigma (St. Louis, USA) unless otherwise stated.
4
Western Blot Analysis of Apoptosis Markers
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Total protein was extracted from the cells subjected to the different treatments using RIPA buffer (Beyotime Institute of Biotechnology) at 4℃. After resolution of the total proteins by electrophoresis, the protein bands were electrotransferred to polyvinylidene difluoride membranes for incubation with primary antibodies against the following target proteins: GAPDH (1:5000 dilution, ab8245, Abcam), caspase‐3 (1:1000, ab4051, Abcam), caspase‐9 (1:1000, ab25758, Abcam), cleaved caspase‐3 (1:1000, ab2302, Abcam), cleaved caspase‐9 (1:1000, ab2324, Abcam), Src homology region 2 domain‐containing phosphatase 1 (SHP‐1) (1:2000, ab227503, Abcam), signal transducer and activator of transcription 3 (STAT3) (1:2500, ab31370, Abcam), and phosphor STAT3 (1:500, ab7315, Abcam). Thereafter, the membranes were incubated with goat anti‐rat secondary antibody (1:1000, A0216, Beyotime Institute of Biotechnology) and horseradish peroxidase‐conjugated goat anti‐rabbit antibody (1:1000; A0208, Beyotime Institute of Biotechnology). The immune response zone was finally detected by development with a microporous chemiluminescence western blot kit (Millipore Sigma).
5
Evaluating GANT61-Cisplatin Cytotoxicity in Cells
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GANT61 purchased from Sigma-Aldrich (St. Louis, MO, USA) was dissolved in DMSO and stored at −20 °C. Cisplatin was obtained from Sigma-Aldrich and was dissolved at a stock concentration of 2 mmol/L. Rhodamine 123 was purchased from Sigma-Aldrich. Foetal bovine serum (FBS) and 0.25 % trypsin/EDTA were bought from Gibco Life Technologies (Carlsbad, CA, USA). A FITC-annexin V kit was purchased from Abcam (Cambridge, MA, USA). The CCK-8 cell count kit for cell proliferation analysis was purchased from Tongren Chemical Research Institute (Kyushu, Japan). The reverse transcription kit was purchased from Takara (Shiga, Japan). SYBR Green I was purchased from Noble Ryder (Beijing, China). Antibodies for GLI1 (ab49314), GLI2 (ab26056), Bax (ab10813), BCL-2 (ab59348), caspase-3 (ab32351) and caspase-9 (ab25758) were purchased from Abcam (Cambridge, MA, USA). The β-actin antibody (AP0060) was purchased from Bioworld (Louis Park, MN, USA).
6
Protein Expression Analysis in HepG2 Cells
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Lysis buffer (200 μl/well) was used to lyse HepG2 cells. Lysis buffer was composed of
Triton X-100 (1%), Tris (50 mM, pH=7.6) and NaCl (150 mM), with inhibitors of phosphatases
and proteases. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%)
was used to separate 40 μg of the total extracted protein. Then Western blotting was done
as demonstrated by Moeschel et al. (10 (link)). Following the application of antibodies for
western blotting anti-PRDX1 (ab109498), anti-beta actin (ab115777), anti
caspase-3 (ab13847 and ab32042), anti-cleave caspase-9 (ab202068 and ab25758), anti-PARP-1
(ab191217), anti-Bim (ab7888), antiFis1 (ab189846), anti-APaf-1 (ab254248), anticytochrome
c (ab133504), anti-Bcl-2 (ab182858), anti-Bax (ab3191), anti-DRP1 (ab184247) and antiDyn2
(ab65556; all purchased from AbCam, UK). Nitrocellulose was blocked using skimmed milk
(5%) or BSA (2%, both from Merck, Germany) for two hours. Subsequently, membranes were
incubated with primary antibodies at 4°C overnight. Before incubation with secondary
antibody, washing was performed (four times for 10 minutes), followed by appropriate
conjugated secondary incubation for one hour. For visualizing expression level of
proteins, enhanced chemiluminescence was performed.
Triton X-100 (1%), Tris (50 mM, pH=7.6) and NaCl (150 mM), with inhibitors of phosphatases
and proteases. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%)
was used to separate 40 μg of the total extracted protein. Then Western blotting was done
as demonstrated by Moeschel et al. (10 (link)). Following the application of antibodies for
western blotting anti-PRDX1 (ab109498), anti-beta actin (ab115777), anti
caspase-3 (ab13847 and ab32042), anti-cleave caspase-9 (ab202068 and ab25758), anti-PARP-1
(ab191217), anti-Bim (ab7888), antiFis1 (ab189846), anti-APaf-1 (ab254248), anticytochrome
c (ab133504), anti-Bcl-2 (ab182858), anti-Bax (ab3191), anti-DRP1 (ab184247) and antiDyn2
(ab65556; all purchased from AbCam, UK). Nitrocellulose was blocked using skimmed milk
(5%) or BSA (2%, both from Merck, Germany) for two hours. Subsequently, membranes were
incubated with primary antibodies at 4°C overnight. Before incubation with secondary
antibody, washing was performed (four times for 10 minutes), followed by appropriate
conjugated secondary incubation for one hour. For visualizing expression level of
proteins, enhanced chemiluminescence was performed.
7
Western Blotting of Apoptosis Markers
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Extracts of cells and specimens were prepared in RIPA buffer (Beyotime Biotechnology) at 4 °C. WB was performed with the cell extracts using commercially available primary antibodies. Primary antibodies used in this study includes: Antibodies to GAPDH (1:5000, ab8245, Abcam), Caspase-3 (1:1000, ab4051, Abcam), Caspase-9 (1:1000, ab25758, Abcam), cleaved Caspase-3 (1:1000, ab2302, Abcam), cleaved Caspase-9 (1:1000, ab2324, Abcam), SHP-1 (1:2000, AB227503, Abcam), STAT3 (1:2500, ab31370, Abcam), and phosphor STAT3 (1:500, ab7315, Abcam). A combination of horseradish peroxidase-conjugated goat anti-rabbit (1:1,000; A0208) and goat anti-mouse secondary antibodies (1:1000; A0216, all from Beyotime Biotechnology) were used to detect the immunoreactive bands; development was performed using the Millipore chromogenic chemiluminescence detection kit (Millipore Sigma, Burlington, MA).
8
Protein Expression Analysis in Cultured Cells
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Whole cell lysate was prepared by lysing cultured cells in radioimmunoprecipitation assay buffer (pH 8.0) containing protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Protein concentrations were determined using a bicinchoninic acid (BCA) kit. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membrane was probed overnight with primary antibodies at 4° C and washed with tris-buffered saline-Tween 20 (TBST). The washed membrane was probed with secondary antibodies for 1 h at room temperature. The following antibodies were used: FASN (ab99359; 270 kDa; 1:500; Abcam, Cambridge, UK), PPAR-γ (ab59256; 54 kDa; 1:100; Abcam), Bax (ab53154; 25 kDa; 1:1000; Abcam), cleaved caspase-9 (ab2324; 40 kDa; 1:1000; Abcam), caspase-9 (ab25758; 50 kDa; 1:1000; Abcam), and Actin (ab8227; 43 kDa; 1:1000; Abcam). After several washes with TBST, the bands on the membrane were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).
9
Antibody Reagent Sources for Apoptosis Analysis
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The antibodies and reagents used in this study were purchased from the following sources: cleaved caspase-9 (ab25758; abcam, Cambridge, MA, USA), cleaved caspase-3 (C8487; Sigma-Aldrich Co., St Louis, MO, USA), PARP (9542; Cell Signaling, Danvers, MA, USA), thioredoxin (abcam, ab26320), GAPDH (10494–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), goat anti-rabbit IgG-horseradish peroxidase (HRP) (ab6721; abcam), goat anti-mouse IgG-HRP (abcam, ab6789), HHT (H0635; Sigma-Aldrich Co.), ETP (E1383; Sigma-Aldrich Co.), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co., D6883), N-acetyl-l -cysteine (NAC) (Sigma-Aldrich Co.; A7250).
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