Caspase activity was quantified using the fluorometric kit Caspase 3 Multiplex Activity Assay (ab219915, Abcam) according to the manufacturer’s protocol. Cells from the PDL cell line PDL26 were grown in a 96-well plate at 2 × 104 cells per well. The cell line was obtained from the third molar of a healthy 26-year-old male non-smoker after written consent according to the ethics regulations of the University of Goettingen (file no.: 27/2/09). Immortalization with human Telomerase Reverse Transcriptase has been described before [64 (link)]. The cells were treated with 10 nM staurosporine (Sigma-Aldrich, Munich, Germany), 60 ng/mL SOD2 (Abcam), 60 ng/mL BIRC3 (Abcam), or staurosporine in combination with SOD2 or BIRC3 for 1 d. Untreated cells served as control. Then, 100 μL/well of caspase assay loading solution was added to each well, and the plate was incubated at room temperature for 60 min and protected from light. Subsequently, the fluorescence intensity was measured using a SpectraMax iD5 Multi-Mode microplate reader (Molecular Devices, CA, USA) at specific wavelengths (Ex/Em = 535/620 nm). For data analysis, the blank readings were subtracted from all measurements, and the CASP3 activity of each group was determined in relation to the control group.
Birc3
Manufactured by Abcam
Sourced in Germany, United Kingdom
BIRC3 is a protein-coding gene that plays a role in the regulation of apoptosis, or programmed cell death. The BIRC3 protein is a member of the baculoviral IAP repeat-containing protein family, which are involved in the inhibition of apoptosis. The specific function of BIRC3 is to inhibit the activity of certain caspase enzymes, which are key mediators of the apoptotic process.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Bcl xl, by Abcam (1 mentions)
Bca protein assay reagent kit, by Thermo Fisher Scientific (1 mentions)
Sds loading buffer, by Beyotime (1 mentions)
Gapdh, by Novus Biologicals (1 mentions)
Staurosporine, by Abcam (1 mentions)
Caspase 3 multiplex activity assay, by Abcam (1 mentions)
Spectramax id5 multi mode microplate reader, by Molecular Devices (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Emd millipore, by Merck Group (1 mentions)
5 protocols using birc3
1
Quantifying Caspase 3 Activity in PDL Cells
2
Cell Lysis and Antibody Sources
3
Western Blotting of BIRC3, NF-κB Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested in the RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and the protein concentration of each sample was determined using BCA protein assay reagent kit (Thermofisher Scientific, Inc) after transfection of 48 h. The supernatants containing total protein were mixed with corresponding volume of 5×SDS loading buffer (Beyotime Biotechnology, Shanghai, China) and heated at 100 °C for 10 min. The lysates of 20 μg were electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The proteins were blocked containing 5% defatted milk for 1 h at room temperature. Subsequently, primary antibodies were incubated at 4 °C overnight, which consisted of the following: BIRC3 (Abcam, cat: ab32059, 1:1000); p-P65 (Abcam, cat: ab6503, 1:1000); P65 (Abcam, cat: ab16502, 1:1000); IκBα (CST, cat: #4812, 1:1000); p-IκBα (CST, cat: #2859, 1:1000); c-myc (CST, cat: 5605, 1:1000); GAPDH (Novus Biologicals, cat: 2D4A7, 1:5,000). Followed by incubation with the horseradish peroxidase conjugated secondary antibodies (anti-rabbit, 1:10,000; cat: #7074; or anti-mouse, 1:10,000; cat: #7076; both from CST, Inc.) for 1.5 h at room temperature. To ensure that equal amounts of sample protein were applied for electrophoresis, GAPDH was used as an internal control.
4
Western Blot Analysis of BIRC3 and GAPDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Data were collected as previously described in previous research (Li et al., 2023 (link)). Specifically, cells were lysed using radio immunoprecipitation assay lysis buffer (Merck KGaA; Merck, Rahway, NJ, USA), and total protein was extracted. Twenty micrograms of protein samples were separated on 10% SDS-PAGE gels, transferred onto PVDF membranes (EMD Millipore; Millipore, Burlington, MA, USA), and blocked at room temperature for 1 h. The membranes were then incubated with primary antibodies (BIRC3 concentration: 0.5 µg/mL, GAPDH dilution rate: 1:500; Abcam; Cambridge, UK) overnight at 4 °C. The following day, the membranes were incubated with a secondary antibody (dilution rate: 1:2,000; Abcam; Cambridge, UK) for 1 h at 24 °C. Signals of the target proteins were detected using an enhanced chemiluminescence detection system. The primary antibodies used in this study were as follows: rabbit polyclonal BIRC3 (24304-1-AP; Proteintech, Wuhan, China) and mouse monoclonal GAPDH (60004-1-Ig; Proteintech, Wuhan, China). These antibodies were meticulously selected for their specificity and reliability in detecting their respective protein targets.
5
Western Blot Analysis of BIRC3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of samples was extracted using RIPA Lysis Buffer (Beyotime, China, which contained PMSF (100:1) according to the standard procedure. The protein samples were loaded onto 10% SDS PAGE and separated. Then, they were transferred to a PVDF membrane. The membrane was blocked for 2 h with 0.5% skim milk powder. The target proteins on the membranes were incubated with the relative primary antibodies β-actin (1:4,000; cat. no. ab8227) and BIRC3 (1:1,000; cat no. ab32059) (both from Abcam, Cambridge, UK) for 2 h on table concentrator at 4°C. After being washed three times, the membranes were sequentially incubated with the HRP conjugated secondary antibody anti-Rabbit IgG H&L (HRP) (1:4,000; cat. no. ab205718) (Abcam) for 1 h at RT and ECL detection followed.
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!