Cultures were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.5% Triton X-100/PBS, and washed 3 times with PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, Adjust to a final pH of 7.4). Primary antibodies were suspended in IF buffer (3% bovine serum albumin, 0.1% NP-40 in PBS) and incubated overnight. The cultures were washed with 0.1% NP-40/PBS 6 times and incubated with Alexa fluorophore conjugated rabbit or mouse secondary antibodies (Life Technologies) in IF buffer. The slides were washed with 0.1% NP-40/PBS 6 times, counterstained with 4', 6-diamidino-2-phenylindole (DAPI), and mounted with antifade solution (Life Technologies). Confocal microscopy was done using the Zeiss LSM 780 laser scanning confocal microscope (Carl Zeiss AG). p-AKT(S473) (4060) and cleaved Caspase-3 (9664) antibodies were from Cell Signaling Technology. Rabbit polyclonal antibody against Ki67 (ab833) was from Abcam, and Mouse anti-laminin-5 (MAB1947) was from EMD Millipore. Alexa Fluor 488 anti-rabbit (A11008) and Alexa Fluor 594 anti-mouse (A21201) secondary antibodies, and ProLong® Gold antifade reagent with DAPI (P36931) were obtained from Life Technologies.
Cleaved caspase 3 9664
Manufactured by Cell Signaling Technology
Sourced in United States
Cleaved caspase-3 (9664) is a primary antibody that detects the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Caspase-3 is a key effector of apoptosis and is activated by both extrinsic (death ligand) and intrinsic (mitochondrial) pathways.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Death receptor antibody sampler kit, by Cell Signaling Technology (2 mentions)
β actin 4970, by Cell Signaling Technology (2 mentions)
Caspase 3 9662, by Cell Signaling Technology (2 mentions)
Gapdh 2118, by Cell Signaling Technology (2 mentions)
Bcl xl 2764, by Cell Signaling Technology (2 mentions)
Bax 2772, by Cell Signaling Technology (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Parp 9542, by Cell Signaling Technology (2 mentions)
Hnrnp e1, by Abnova (2 mentions)
Hsp90 sc 13119, by Santa Cruz Biotechnology (2 mentions)
Ab24359, by Abcam (2 mentions)
E cadherin 3195, by Abcam (2 mentions)
Ab102470, by Abcam (2 mentions)
Arf4 11673 1 ap, by Proteintech (2 mentions)
Bbf2h7 creb3l2 mabe1018, by Merck Group (2 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by GE Healthcare (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Ecl plus chemiluminescence kit, by GE Healthcare (2 mentions)
Cleaved parp 5625, by Cell Signaling Technology (2 mentions)
27 protocols using cleaved caspase 3 9664
1
Immunofluorescence Staining of Cell Cultures
2
Protein Extraction and Western Blot Analysis
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Total protein from tissues and cell lines was extracted using RIPA (Thermo Scientific, Rockford, IL, USA), and nuclear protein was prepared using the Nucleoprotein Extraction Kit (Sangon Biotech, Shanghai, China) in the presence of protease inhibitor cocktail and PMSF (Beyotime Institute of Biotechnology, Haimen, China). The concentration of various protein samples was determined using the BCA Protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Thirty micrograms of each lysate was electrophoresed in 10% SDS-PAGE gels and then transferred onto PVDF membranes and detected using the ECL kit (Beyotime Institute of Biotechnology). The SETD2 (ab69836), p53 (ab28) and H3K36me3 (ab9050) antibodies were purchased from Abcam (Cambridge, MA, USA). GAPDH (D16H11), Caspase-3 (#9662) and cleaved Caspase-3 (#9664) antibodies were obtained from Cell Signaling Technology Inc. (Vebery, MA, USA). H3 antibody was ordered from Beyotime Institute of Biotechnology. Anti-rabbit and anti-mouse IgG secondary antibodies conjugated with horseradish peroxidase were provided by Wuhan Boster Bio-engineering Limited Company (Wuhan, China). ImageJ Software version 1.36b was applied to analyze the expression levels of different proteins.
3
Immunoblotting Analysis of Cellular Fractions
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The nucleus and cytoplasmic fractions of CCA cells were prepared as previously described25 (link). Cell lysates of the entire cells or nucleus fractions were collected by digesting with RIPA buffer and protease inhibitors (Sigma-Aldrich, USA). Protein concentration was determined by BCA kit (Thermo Fisher, USA). A total of 30 μg protein was loaded on 10% or 15% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were incubated with specific first antibodies and corresponding second antibody. The specific antibodies were listed below: Cleaved Caspase-3 #9664, PCNA #13110; MMP-3 #14351, ZEB1 #70512, E-Cadherin #3195, Vimentin #5741, Oct-4 #2750, LIN28A #3695, Nanog #3580, Sox2 #3579, β-Catenin #8480, Phospho-β-Catenin (Ser33/37/Thr41) # 9561, Phospho-GSK-3β (Ser9) #5558, GSK-3β#12456, c-Myc #9402, Survivin #2803, Lamin B1 #12586, and GAPDH #5174 were purchased from Cell Signaling Technology with a work concentration at 1: 1000. BCL9L #ab233736 was purchased from Abcam with a work concentration at 1: 500. The second antibodies were goat anti-rabbit IgG HRP-linked antibody (Cell signaling #7074; 1:4000) and sheep anti-mouse IgG-HRP (GE/Amershan #NXA931; 1: 5000).
4
Quantification of Protein Expression
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Primary antibodies (TRX #2429, p-ASK1 #3765, p-JNK1/2 #9251, JNK1/2 #9252, Cleaved Caspase-3 #9664, HA-Tag #3724, GAPDH #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies (Alexa Fluor® 488 anti-rabbit #A-11034 and anti-rabbit #sc-2004) were purchased from Invitrogen (Carlsbad, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. CONPs were purchased from the NanoScale Corporation (Manhattan, KS, USA) or synthesized as previously described [14 (link),36 (link)]. For western blotting, cells were plated in 10 cm dishes and left to attach overnight. Media was then removed and replaced with media containing 0 or 10 µM CONPs. 24 h later, cells were exposed to 0 or 5 Gy RT with cell lysates collected 24–72 h after radiation. Proteins were separated on 12.5% SDS-PAGE gels, transferred to nitrocellulose membranes using the iBlot system (Invitrogen, Carlsbad, CA, USA), and blotted following standard procedures. The chemiluminescence in the blots were recorded and quantified by selecting the same exposure duration for all the membranes using Image Lab 3.0 (Bio-Rad, Hercules, CA, USA) as described previously [35 (link)].
5
Apoptosis Signaling Pathway Analysis
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The following antibodies and reagents were used: Sirtuin Antibody Sampler Kit #9787T, NF‐κB p65 Antibody Sampler Kit #4767, Death Receptor Antibody Sampler Kit #8356, antibodies against cleaved PARP #5626, cleaved caspase‐3 #9664, cleaved caspase‐8 #8592, cleaved caspase‐9 #9509, Bak #6947, Bax #5023, Bcl‐2 #15071, and Bcl‐X #2764 (Cell Signaling Technology, Danvers, MA, USA); antibodies against HDAC3 #sc‐130319 and β‐actin #sc‐58673 (Santa Cruz Biotechnology, Dallas, TX, USA); nicotinamide (NAM); and ammonium pyrrolidine dithiocarbamate (PDTC) (Sigma‐Aldrich, St Louis, MO, USA).
6
Western Blot Analysis of Cell Signaling
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Cells lysates were collected using Laemmli buffer (0.05 M Tris-HCl at pH 6.8, 1% SDS, 10% glycerol, 0.1% β-mercaptoethanol), quantified using BCA quantification (Thermo Fisher Scientific) and resolved using Criterion XT pre-cast gels (BioRad) followed by transfer to PVDF membranes. Antibodies used are as follows: pS6S240/244 #5264, S6 #2317, p53 #2524, p4E-BP1T37/46 #2855, total 4E-BP1 #9644, cleaved caspase-3 #9664, cleaved PARP #9544, pAKTT473 #4060, TSC2 #4308, β-actin #4970; all from Cell Signaling Technology and used at 1:1000 dilutions. All uncropped western blots can be found in Supplementary Fig. 9 .
7
Investigating ABCC2 and Apoptosis in A549 Cells
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A549 and A549/DDP cells were lysed on ice for 30 minutes in lysate buffer (RIPA; Beyotime, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (1 mM). The lysates were centrifuged at 12000 rpm at 4°C for 15 minutes. The supernatant was collected, and protein concentrations were determined using a bicinchoninic acid assay (KeyGen Biotech, Jiangsu, China). The supernatant was mixed with 5 × SDS at a 4:1 ratio and incubated at 100°C for 10 minutes. The proteins (20 μg) were then separated on SDS‐polyacrylamide gels (12% separation gels) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, Massachusetts). The PVDF membranes were blocked with 5% skim milk (skim milk dissolved in TBST) for 2 hours at room temperature. The primary antibody was ABCC2 (ab205718, 1:1000) antibody purchased from Abcam. Caspase‐3 #9662, PARP #9532, Cleaved Caspase‐3 #9664, and β‐actin #4970 were purchased from Cell Signalling Technology (dilution 1:1000).
8
Immunofluorescence analysis of endothelial cell markers
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The hMVECs in the microfluidic chip were fixed with 4% paraformaldehyde (PFA) at 25 °C for 30 min. Then, the cells were permeabilized with 0.1% Triton X-100 for 15 min at RT. In sequence, the cells were blocked with 3% bovine albumin serum (BSA) for 1 h at RT. Then, 50 µL of the diluted primary antibodies in the PBS solutions were added to the central channel and stored at 4 °C overnight. Secondary antibodies were conjugated with primary antibodies at RT for 1 h. All the primary and secondary antibodies are listed in Table 1 . In each step, two sets of PBS washes were included. The fluorescence images were captured with a confocal laser-scanning microscope (LSM700; Carl Zeiss, Germany). Actin fibers were evaluated in total 45 randomly chosen hMVECs for each radiation doses and cells were divided into three groups differing in the number of actin stress fibers (0, 1–10, and > 10). The average intensity of VE-cadherin on cytoplasm and cell–cell border was measured by using Image J software program.
Primary and secondary antibodies.
| Marker | Cat.no. (company) | Dilution ratio |
|---|---|---|
| Anti-VE-cadherin | ab33168 (abcam) | 1:200 |
| Anti-Claudin 5 | ab15106 (abcam) | 1:200 |
| Anti-DLL4 | ab7280 (abcam) | 1:1000 |
| Anti-Cleaved Caspase 3 | 9664 (Cell signaling) | 1:400 |
| Anti-Cleaved Caspase 9 | 20750 (Cell signaling) | 1:400 |
| Goat anti-rabbit IgG, Alexa fluor 488 | A-11008 (Invitrogen) | 1:200 |
9
Investigating Hypoxia Signaling Pathways
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2-MeOE2 (S1233) and DMOG (S7483) were purchased from Selleck (China). Percoll (17089101) was purchased from GE Healthcare (United States). Collagenase IV (C8160) was purchased from Solarbio (China). Mouse monoclonal antibody for β-actin (sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). A rabbit monoclonal antibody for IL-1ra (ab124962) was purchased from Abcam (Cambridge, MA, United States). Rabbit antibodies for HIF-1α (36169), BAX (14796), Bcl-2 (3498), and cleaved caspase-3 (9664) were purchased from Cell Signaling Technology (Danvers, MA, United States). ELISA kits for detecting mouse alanine transaminase (ALT) (JL12668), aspartate transaminase (AST) (JL13793), and IL-1ra (JL20255) were purchased from Jonln (China). Recombinant IL-1ra (200-01RA) was purchased from PeproTech (United States).
10
Quantification of Apoptosis-Related Proteins
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The total protein from the brain tissue or cells was extracted and quantified using the bicinchoninic acid method. The equivalent weight of the proteins (40 μg/lane) was separated on a 10% SDS‐PAGE gel and was then transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% non‐fat milk in TBST buffer for 1 hour and then incubated overnight with the following primary antibodies: Bax, 2772 (Cell Signalling Technology); Bcl‐2, ab32124 (Cell Signalling Technology); Bcl‐xl, 2764 (Cell Signalling Technology); cleaved caspase‐3, 9664 (Cell Signalling Technology); GAPDH, 2118 (Cell Signalling Technology); and TIM‐4, ab47637 (Abcam). After being washed twice with TBST, the membranes were incubated with a horseradish peroxidase‐conjugated secondary antibody (anti‐rabbit‐HPR; 7074; Cell Signalling Technology) at a 1:2000 dilution. Specific bands were visualized using an enhanced chemiluminescence detection kit.
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