Cells were treated with VRB for 24h in 6-well plates (for western blot analysis of total/Cleaved Caspase 3, 2×105 cells) or on cover slips (for immunofluorescence with detection of Cleaved Caspase 3, 105 cells). The total/cleaved detection of Caspase 3 in western blot was done using Caspase 3 total (Cell Signaling, #9662) and Cleaved Caspase 3 fragment in immunofluorescence was done using Cleaved Caspase 3 (Cell Signaling, #9664).
Total caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Total caspase-3 is a laboratory reagent used to detect and quantify the presence of caspase-3, a key enzyme involved in the process of apoptosis, or programmed cell death. This product provides a reliable and accurate method for measuring the total levels of caspase-3 in cell or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (24 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Cleaved parp, by Cell Signaling Technology (4 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
β actin, by Abcam (3 mentions)
Phospho chk2, by Cell Signaling Technology (2 mentions)
γh2ax, by Cell Signaling Technology (2 mentions)
Species specific horseradish peroxidase conjugated secondary antibodies, by Santa Cruz Biotechnology (2 mentions)
Total caspase 9, by Cell Signaling Technology (2 mentions)
Phospho chk1 ser296, by Cell Signaling Technology (2 mentions)
Total chk1, by Cell Signaling Technology (2 mentions)
Total chk2, by Cell Signaling Technology (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
31 protocols using total caspase 3
1
Evaluating Apoptosis Induction by VRB
2
Western Blot Analysis of Protein Signaling
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Cells were cultured in 100 mm dishes or 6-well plate and harvested in radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). Proteins were separated on SurePAGE (M00654; GenScript) gel by electrophoresis and transferred to polyvinylidene difluoride membrane. Membranes were blocked for 1 h in 5% nonfat dry milk in Tris-buffered saline (TBS) before overnight incubation with diluted primary antibody. The membrane was then washed with TBS and incubated for 1 h with either rabbit or mouse IgG horseradish peroxidase–conjugated secondary antibody (R&D systems). After multiple washing steps, the signal was detected using Amersham ECL Select Western Blotting Detection Reagent and bands were visualized by Amersham Imager 680 (GE healthcare). Primary antibodies used in this article include anti-Prx4 (ab184167; Abcam, 1:10,000) and anti-β-actin (A2228; Sigma–Aldrich, 1: 5000). Antibodies for phosphorylated GSK3α/β (S9/S21), total GSK3α/β, phosphorylated Akt (T308), phosphorylated Akt (S473), Akt (total), phosphorylated PDK, total PDK (S241), phosphorylated PP2A (Y307), PP2A, cleaved caspase 3, and caspase 3 (total) were from Cell Signaling Technology using 1:1000 dilution.
3
Exosomal Protein Analysis by Western Blot
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Western blotting of exosomes was performed as previously described 23 (link). Tissue samples were collected from the ipsilateral hemisphere and quantified with the BCA protein assay (Pierce, Rockford, IL, USA).The primary antibodies were those targeting CD9 (1:1000), TSG101 (1:500), ARG (1:200), CD206 (1:1000), total caspase-3 (1:1000, Cell Signaling technology, Beverly, MA), cleaved caspase-3 (1:1000), USP14 (1:1000 dilution, Abcam), and β-actin (1:1000). For quantify western blot data, we analyzed the band gray values of 4 samples. After calculating the gray value of the band relative to the expression of the internal reference, we standardized the levels to those of the control group and then performed statistical analysis.
4
Immunoblotting Analyses of Liver Proteins
5
Immunoblotting Analysis of Apoptotic Markers
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Protein was analyzed by SDS-PAGE as previously described (16 (link)). The following primary antibodies from Cell Signaling Technology were used at manufacturer-recommended dilutions for immunoblotting: cleaved caspase-3 (#9661), total caspase 3 (#9668), cleaved caspase-9 (#9501), total caspase-9 (#9502), phospho-Chk1 (Ser296; #2349), total Chk1 (#2360), phos-pho-Chk2 (Thr68; #2661), total Chk2 (2662), and γH2AX (#9718). β-Actin (Santa Cruz Biotechnology, catalog #sc-47778) was included as a loading control. Species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used at 1:20,000 dilution.
6
Immunoblotting Analysis of Apoptotic Markers
7
Western Blot Analysis of Cell Signaling
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Whole cell lysates were prepared with lysis buffer. Extracts were resolved on SDS-PAGE, and transferred to nitrocellulose membrane (Whatman, Scheicher & Schuell, Dassel, Germany). Membranes were incubated with the specific antibodies overnight at 4°C, washed and incubated with the appropriate secondary antibody, for 1 h at room temperature. Antibodies were used against: pAKT (s473) #9271, pS6R (s235/236) #2211, pMEK (s217/221) #9121, LC3B(D11) #3868, pBRAF #2996, total Caspase-3 #9662, cleaved caspase-3 #9661 from Cell Signalling (Danvers, MA, USA), BECN1 (sc-10086), SQSTM1/p62 (sc-28359), PARP (sc-7150), pERK (sc-7583), and Tubulin (sc-8035) from Santa Cruz (Biotechnology, Inc. 2145 Delaware Avenue Santa Cruz, CA 95060 USA). Antibody signal was obtained with the enhanced chemiluminescence plus Western blotting detection system (Amersham Biosciences, Uppsala, Sweden) after exposure to Kodak Super RX film. Values were measured using the Image-Quant software (Amersham Biosciences) and protein levels were normalized against tubulin. Experiments were independently repeated three times and standard deviation is presented.
8
Western Blot Analysis of Rg3 Signaling
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Western blot analysis was performed as previously described [19 (link)]. Briefly, MPBAs were treated with the indicated doses of Rg3 for two days. Total proteins were extracted using lysis buffer containing protease and phosphatase inhibitors, followed by processing for SDS-PAGE. The blots were incubated with the following primary antibodies as appropriate: PPARγ, perilipin, ERO1L, Phospho-eIF2α, CHOP, Phospho-Akt, total Akt, cleaved caspase3, total caspase3, and β-actin (all antibodies purchased from Cell Signaling Technology). An ECL Prime Western Blotting System (GE Healthcare) and ImageQuant LAS4000 (GE Healthcare) were used to detect protein bands. Densitometry analysis was performed using Image J software (NIH, Bethesda, MD, USA).
9
Western Blot Analysis of Protein Markers in MIN6 Cells
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Total protein from MIN6 cells was prepared using radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with protease and phosphatase inhibitors. The proteins were separated in 4% to 12% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to polyvinylidene difluoride membranes. Several proteins were detected and measured with antibodies for S1P, phosphor-stress activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) (T183/Y185), SAPK/JNK, cleaved caspase-3 (D175), total caspase-3, β-actin (Cell Signaling Technology, Danvers, MA, USA), pancreatic and duodenal homeobox 1 (Pdx-1, Millipore, Temecula, CA, USA), lamin B1, mitofusin 1 (MFN1, Abcam, Cambridge, MA, USA), mitochondrial dynamin-like GTPase (OPA1, BD Biosciences, San Diego, CA, USA), oxidative phosphorylation (OxPHOS) complex cocktail (Invitrogen, Rockford, IL, USA), and PHB (Santa Cruz Biotechnology).
10
Western Blot Analysis of Stress Response Proteins
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Cellular proteins were prepared by a RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with protease and phosphatase inhibitors. The concentration of protein was measured by the Bradford assay (Amersham Life Science, Arlington Height, IL, USA), and 20 µg of protein from each group was separated in 4% to 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels and transferred to polyvinylidene difluoride membranes. Proteins in the membranes were probed with primary antibodies for p-eIF2α (Ser51), IRE1α (14C10), CHOP (L63F7), phosphor-NF-κB p65 (S536), NF-κB, phosphor-stress-activated protein kinase (SAPK)/JNK (T183/Y185), SAPK/JNK, cleaved caspase-3 (D175), total caspase-3, β-actin (Cell Signaling Technology, Danvers, MA, USA), ATF6 (Abcam, Cambridge, MA, USA), and GRP78 (Santa Cruz Biotechnology) for 24 hours. The membranes were then exposed to secondary antibodies conjugated with horseradish peroxidase, and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL, USA). ImageJ, a program for quantification, was used to measure the density of the scanned immunoblot bands. The density of phosphorylated NF-κB, SAPK/JNK, and cleaved caspase-3 was normalized to the levels of NF-κB, SAPK/JNK, and caspase-3, respectively.
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