hucMSC-exosomes or H9C2(2-1) cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and phenylmethanesulfonyl fluoride (PMSF). The protein concentration was determined using the BCA protein assay kit. Equal quantities of protein were loaded and run on 12% SDS gels and then transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk for 1 h and incubated with diluted primary antibodies [CD9 (1 : 500; Bioworld), CD63 (1 : 300; SAB), Bcl-2 (1 : 500; Bioworld), Bax (1 : 500; Bioworld), and GAPDH (1 : 2,000; CWBIO)] at 4°C overnight. The membranes were incubated in goat anti-rabbit or anti-mouse antibodies (1 : 2,000; Bioworld) at 37°C for 1 h. The target proteins were detected using the Luminata Crescendo Western HRP substrate (Millipore, USA) and the results were analyzed by AlphaView SA.
Bcl 2
Manufactured by Bioworld Technology
Sourced in United States, China
Bcl-2 is a lab equipment product that serves as a key regulator of apoptosis, or programmed cell death. It functions to inhibit the activation of caspases, which are enzymes that play a central role in the execution of the apoptotic process. Bcl-2 is widely used in various areas of biological research to study cell survival and death mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Bioworld Technology (17 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Caspase 3, by Bioworld Technology (4 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
Gapdh, by Bioworld Technology (4 mentions)
Caspase 3, by Santa Cruz Biotechnology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
β actin, by Bioworld Technology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Luminata crescendo western hrp substrate, by Merck Group (2 mentions)
Cytochrome c, by Cell Signaling Technology (2 mentions)
Image system, by Bio-Rad (2 mentions)
Gapdh, by CWBIO (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Erk1 2, by Bioworld Technology (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
38 protocols using bcl 2
1
Western Blot Analysis of Exosomal Markers and Apoptotic Proteins
2
Immunoblotting of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the immunoblotting of P53, Bcl-2, Bax, and caspase-3, proteins were extracted from whole cell lysates using RIPA buffer (Cell Signaling) following manufacturer's instructions. For the immunoblotting of Cytochrome c and Smac, cytoplasmic proteins were extracted using NE-PER Nuclear Protein Extraction Kit (Thermo Fisher). The proteins separated by SDS gel were transferred to 0.22 μm PVDF membrane (Bio-Rad) at 15 V for 2 h by using semidry transfer set (CBS Scientific). After blocking the membranes with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) (150 mM NaCl, 15 mM Tris-HCl (pH 7.5), and 0.1% Tween 20) for about 2 h, proteins were probed with primary antibody against Bcl-2 (Bioworld), caspase-3 (Bioworld), P53 (Bioworld), Bax (Bioworld), Smac/Diablo (Cell Signaling), Cytochrome c (Cell Signaling), or β-actin antibody (Santa Cruz) and then incubated with a secondary antibody conjugated with horseradish peroxidase (Cell Signaling). Membranes were washed by TBST after each antibody probing. Signals were detected by using Super Signal West Pico chemiluminescent substrate (Thermo Scientific). Integrated optical density (IOD) quantification was performed using Gel-Pro Analyzer 4.0.
3
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissues were washed with cold PBS and lysed in ice-cold RIPA buffer containing protease inhibitors (Beyotime, China), followed by incubation on ice for 30 min and centrifugation (4°C, 30 min). The supernatant was collected, and protein concentration was measured. Equal amounts of protein from each sample were separated by electrophoresis on a 10% SDS-polyacrylamide gel (Beyotime, China), electrotransferred to a PVDF membrane (Millipore, USA) and blocked. Antibodies against GAPDH (Santa Cruz, 1:1000), Bcl-2 (Bioworld, 1:500), and Wnt2 (Abcam, 1:1000) were used. Immunoblots were visualized by chemiluminescence using an ECL detection system (BeyoECLPlus, Beyotime, China). The intensity of the bands was determined using Image Pro Plus 6.0 software.
4
Western Blotting Analysis of Histone Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six mice of NaB- and Veh-treated groups were used for western blotting. Mice were killed by cervical dislocation and the cortex and the hippocampus were quickly dissected. Histone protein and total protein were prepared from both tissues as previously described, respectively (Shechter et al., 2007 (link); Li et al., 2011 (link)). Purified proteins were separated on SDS-PAGE and quantified using Odyssey LI-COR. GAPDH (1:5000, Bioworld, St. Louis Park, MN, USA) was used as loading control. Commercial primary antibodies used include Acetylated Histone 3 (Ac-H3; 1:2000, Upstate Biotechnology, Merck Millipore, Darmstadt, Germany), Histone 4 (1:1000, Bioworld, St. Louis Park, MN, USA), Tau-P (phospho S199 + S202; 1:1000, Abcam, Cambridge, UK), Caspase 3 (1:500, Bioworld, St. Louis Park, MN, USA), cleaved Caspase 3(1:100, chemicon, Merck Millipore, Darmstadt, Germany), Bcl-2 (1:500, Bioworld, St. Louis Park, MN, USA) and glial fibrillary acidic protein (GFAP; 1:500, ZSGB-Bio, Beijing, China). Secondary antibodies used included anti-Mouse IRDye 800CW and anti-Rabbit IRDye 800CW (1:5000, Li-Cor Biosciences, Cambridge, UK).
5
ZnO Nanoparticle Cytotoxicity Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ZnO NPs were obtained from HT Nano Company (Nanjing, China). Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12) was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from PAA (Pasching, Austria). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC), and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). SP600125 and SB203580 were obtained from Beyotime (Shanghai, China). U0126 was obtained from Promega (Madison, WI, USA). Bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL, USA). Annexin V-FITC apoptosis kit was purchased from Abcam (Mountain View, CA, USA). Bcl-2, Bax, JNK, ERK1/2, p38 MAPK, phosphor-JNK, phosphor-ERK1/2, phosphor-p38, and caspase-3 antibodies were purchased from Bioworld (St. Louis Park, MN, USA); poly(ADP-ribose) polymerase-1 (PARP) was purchased from Cell Signaling Technology (Boston, MA, USA); β-actin and secondary antibodies (goat anti-mouse or anti-rabbit IgG-conjugated horseradish peroxidase (HRP)) were purchased from Beyotime (Shanghai, China).
6
Selective DOR Agonist and Autophagy Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
[d -Ala2, d -Leu5]-Enkephalin (DADLE, a selective DOR agonist), 3-methyladenine (3-MA, an autophagy inhibitor) and antibody against LC3 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Naltrindole (a DOR antagonist) was obtained from Tocris (Ellisville, MO, USA). DMEM, FBS, 0.05% Trypsin-EDTA and normal goat serum were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) kit, LDH kit, BCA kit, Cyanine 3 (Cy3)-conjugated secondary anti-rabbit antibodies, Alexa Fluor 488 goat anti-mouse IgG and Hoechst were obtained from Beyotime (Nantong, Jiangsu, China). PMSF (a protease inhibitor cocktail and a phosphatase inhibitor cocktail) was obtained from KangChen (Shanghai, China). Antibodies against beclin 1, p62 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against glial fibrillary acidic protein (GFAP), Bcl-2, Bax and an anti-rabbit IgG antibody linked with HRP were obtained from Bioworld (Shanghai, China). An ECL kit was obtained from Millipore (Billerica, MA, USA).
7
Quantitative Analysis of Apoptosis-Related Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated from heart tissue with a RIPA buffer, and the protein concentration was measured with a BCA assay kit. The protein samples were separated by SDS-polyacrylamide gels with suitable concentration and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bull serum albumin and then developed with diluted antibodies for Bax (14796, 1:1000, Cell Signaling Technology, Beverly, MA, USA), Bcl-2 (BS1511, 1:1000, Bioworld, Bloomington, USA), Cleaved Caspase-3 (9661, 1:1000, Cell Signaling Technology, Beverly, MA, USA), METTL3 (ab240595, 1:1000, Abcam, Cambridge, MA, USA), FTO (27226-1-AP, 1:1000, Proteintech, Wuhan, China), YTHDF1 (17479–1-AP, 1:1000, Proteintech, Wuhan, China), and GAPDH (10494-1-AP, 1:5000, Proteintech, Wuhan, China) overnight at 4 °C. Subsequently, membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody at room temperature for 90 min. The membranes were observed on Image Systems (Bio-Rad, Hercules, CA, USA). Image Lab software was used for semiquantitative calculation.
8
Hippocampal Apoptosis Markers Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The right hippocampus was assigned to detect the protein expression of Caspase-3 (CASP3), B-cell lymphoma 2 (BCL2), and BCL2-associated X protein (BAX) using Western blotting assays (n=6 for each group). For protein extraction, tissue was collected and added to RIPA buffer with 1% PMSF and then homogenized in lysis buffer on ice. Total proteins were extracted following the manufacturer’s instructions (Applygen Technologies, Inc., China). After determining protein concentrations, samples (30–50 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to immobile polyvinylidene difluoride membranes. Nonspecific bindings were blocked with 5% nonfat milk in TBST buffer (pH=7.4) for 1 h, then the membranes were incubated with the following primary antibodies overnight at 4°C: anti-CASP3 (1: 500; Bioworld, USA), BCL2 (1: 500; Bioworld, USA), BAX (1: 500; Bioworld, USA), and anti-β-actin (1: 1000; Santa Cruz, CA, USA). Subsequently, after washing with 0.1% TBST, membranes were incubated with secondary antibody (1: 4000; Rockland, Gilbertsville) for 1 h at room temperature and washed in 0.1% TBST solution. Bands were detected using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) and the relative density of bands was estimated using Image J analysis software.
9
Protein Expression Analysis of Exosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells, exosomes, and tissues were lysed in RIPA buffer. Protein concentration was determined using the BCA assay kit (Pierce, USA). Sources and dilution factors of primary antibodies were: rabbit polyclonal CD63 (1:1000; Bioworld, USA), CD9 (1:1000; Bioworld), CD81 (1:1000; Epitomics, USA), PCNA (1:1000; Bioworld), BCL-XL (1:100; SAB, USA), BCL-2 (1:1000; Bioworld), Bax (1:1000; Bioworld), Cytochrome C (1:500; Abcam, USA), caspase-3 (1:500; Bioworld), IL-1β (1:500; Bioworld,), LC3B (1:500; Abcam), Beclin-1 (1:600; Proteintech, USA), mTOR (1:500; SAB), p-mTOR (1:500; SAB), 4EBP1 (1:200; SAB), p70S6K (1:200; SAB), and mouse monoclonal GAPDH (1:3000; Kang Chen, China). The nucleoprotein and plasma protein was separated by the nuclear and plasma protein isolation kit (Vazyme, China), with the primary antibody NF-kB-P65 in the nucleus (1:500; SAB), primary antibody nucleoprotein Histone (1:1000; SAB). After incubation with the primary antibodies overnight at 4 °C, membranes were washed three times with Tris-buffered saline with 0.05% Tween-20 and challenged with HRP-conjugated goat anti-rabbit or goat anti-mouse antibody (1:2000; Bioworld). Western blot was performed by Luminata™ crescendo western HRP substrate (Millipore, USA) and analyzed using MD Image Quant Software.
10
Western Blot Analysis of Cardiac Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cardiac tissues and transfected cells were homogenized in RIPA buffer (Heart, Beijing, China). BCA Protein Assay Kit was used to determine the protein concentrations. Then, 50 μg of total protein per sample was subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a PVDF membrane (BioRad, Hercules, CA, USA). The membrane was then incubated with the primary antibody of interest, washed with PBS-T, and incubated with an appropriate secondary antibody. Protein bands were quantified with ImageQuant software. The β-actin level was used as an internal control. The following primary antibodies were used: ADAR1 (Santa Cruz Biotechnology, Dallas, TX, USA), Dicer (Biorbyt, Cambridge, UK), PTEN (Abcam, Cambridge, UK), BAX (Cell Signaling Technology, Danvers, MA, USA); Bcl-2 (Bioworld, Nanjing, China); β-actin (Cmctag, Shanghai, China); GAPDH (Cmctag, Shanghai, China). Three independent experiments were performed for each sample.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!