The experiment was performed as described previously (Padder et al., 2021 (link)). Briefly, H1299 and A549 cells were treated with different concentrations of 18α-GA for 48 h, trypsinized, and lysed in RIPA lysis buffer supplemented with 1x Phos stop and Sigma fast (Sigma Aldrich) for 10–15 min on ice. The lysates were centrifuged at 10,000 rpm for 10 min at 4°C; the supernatant was taken and total protein was quantified using a BCA kit (Thermo Scientific). Equal amounts of protein (20–50 μg) were resolved on SDS-PAGE gels, followed by transfer of the resolved proteins onto PVDF membrane (Bio-Rad) using a wet transfer system at 60 V–80 V for 1 h. The membranes were then blocked with 5% non-fat milk in TBST for 40 min and probed overnight with indicated primary antibodies at 4°C and later probed with appropriate HRP conjugated secondary antibodies at room temperature for 1 h. The protein bands were then developed onto x-ray film (kodak) and the ChemiDoc imaging system. The primary antibodies used were against, beta-actin, caspase-3, Bax, and Bcl2 (Novus Biologicals), EGFR and pPKBa (Cloud Clone), pPI3K (CST), and the corresponding secondary antibodies used were either goat anti-rabbit IgGHRP (Novus Biologicals) or goat anti-mouse IgGHRP, (Novus Biologicals).
Bcl2 associated x (bax)
Manufactured by Novus Biologicals
Sourced in United States
Bax is a lab equipment product that is used in various research applications. It serves as a core function in the analysis and study of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Novus Biologicals (5 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Western blot detection kit, by Bio-Rad (2 mentions)
Anti rat igg, by Thermo Fisher Scientific (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
β actin, by Novus Biologicals (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Caspase 3, by Novus Biologicals (1 mentions)
Sigmafast, by Merck Group (1 mentions)
Phosstop, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Caspases 3, by Cell Signaling Technology (1 mentions)
Caspase 8, by Novus Biologicals (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Penicillin streptomycin mixed, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
8 protocols using bcl2 associated x (bax)
1
Evaluating 18α-GA's Impact on Apoptosis Signaling
2
Immunoblot Analysis of Apoptotic Proteins
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Immunoblot analysis was used to investigate cell apoptotic signal proteins. RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) was used to extract total proteins, and the concentration of proteins were then measured using the Bradford assay. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were separated and then transferred to a polyvinylidene fluoride membrane (Amersham Bioscience, Uppsala, Sweden). The membranes were incubated with primary antibody, including anti-human poly (ADP-ribose) polymerase (PARP, #9542 s; Cell Signalling, Danvers, MA), caspases-3 (#9665 s; Cell Signalling), caspase-9 (#9508 s; Cell Signalling), bax (NB100–56095; Novus Biologicals, Centennial, CO), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-32233; Santa Cruz), at 4 °C overnight. After vigorous washing with tris-buffered saline, the horseradish peroxidase-conjugated secondary antibodies, such as anti-rabbit IgG or anti-rat IgG (Invitrogen, Waltham, MA), were applied to the membranes at room temperature for 1 hrs. The specific bands were developed using the ImageQuant LAS 4000 chemiluminescence imaging equipment (GE Healthcare, Chicago, IL) and a western blot detection kit from Bio-Rad (Hercules, CA).
3
Histopathological Analysis of Kidney Tissues
4
Cytotoxicity and Apoptosis Assessment of 18α-GA
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High-glucose Dulbecco’s Modified Eagles Medium (DMEM), Fetal Bovine Serum (FBS), and the penicillin-streptomycin mixed solution were procured from Gibco-life technologies, Thermo Fischer Scientific (USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), propidium iodide (PI), DAPI, Acridine orange were acquired from Sigma. Bax, β-actin, and Bcl-2 primary antibodies were purchased from Novus Biologicals (Centennial, CO, USA). FITC/Annexin V apoptosis detection kit was purchased from BD Bioscience. The compound 18α-GA was purchased from Sigma-Aldrich with purity ≥95% and stock solutions were dissolved in DMSO and stored at −20°C. All other reagents were of analytical grade and purchased from local suppliers. All the experiments were repeated at least three times.
5
Histological and Immunohistochemical Analysis of Colon Carcinogenesis
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Immediately after mice were euthanized, intestines were harvested in and placed in 10% formalin overnight and then processed for embedding in paraffin. Paraffin‐embedded sections (5 μm) were treated with xylene; rehydrated in decreased ethanol series, and stained with hematoxylin and eosin (H and E) or immunohistochemistry and examined under a light microscope. To access the severity of AOM‐DSS‐induced colon carcinogenesis, the intensity of apoptotic and inflammatory markers was determined using IHC analysis. Colon and other intestinal tissues were immune stained with Caspase‐3 (Abcam, Cambridge MA), Bax (Novus, Littleton, CO), iNOS (Abcam), and COX‐2 (Abcam) as previously described and examined under a light microscope. Colon and other intestinal tissues were also stained with 1:200 dilution of anti‐KI67 (Abcam) and later with 1:200 dilution of anti‐rabbit secondary antibody (Abcam). The sections were then counterstained with hematoxylin and finally dehydrated and covered with coverslips.
6
Protein Expression Quantification Protocol
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The protein expression levels were determined by staining with primary antibodies and relevant HRP conjugated secondary (1:5000, Armersham, Buckinghamshire, UK) antibodies. The primary antibodies (Santa Cruz, CA, USA: Bcl2, Bax, p65, IκBα, nucleolin, ALDH1A1, 1A3, 3A1, 2 and cleaved PARP; Novus Bio, Cambridge, UK: HIF2; Abcam, Cambridge, UK: phosphorylated p65_S276; Cell Signaling, Herts, UK: AKT, phosphorylated AKT, Sox2, Oct4, JNK, phosphorylated JNK, cJun, phosphorylated cJun, phosphorylated p38, ERK) were diluted at 1:1000 in 5% fat-free milk-TBST. Anti-α-tubulin (1:8000, Sigma) and nucleolin were used as a loading control. The signal was detected using an ECL Western blotting detection kit (GeneFlow, Staffordshire, UK).
7
Protein Extraction and Western Blot Analysis
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For protein analysis, total proteins were extracted using RIPA buffer and 1% NP-40 lysis buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The extracted proteins were quantified using the Bradford assay and subjected to immunoblot analysis. Briefly, the proteins were separated using SDS-PAGE and transferred to a PVDF membrane (Amersham Bioscience, Uppsala, Sweden). The first antibody, anti-human PARP (#9542s; Cell Signalling), Caspase-3 (#9665s, Cell Signalling), Caspase-9 (#9508s; Cell Signalling), Bax (NB100-56095; Novusbio), Bcl-2 (NB100-56098; Novusbio), PDHA (sc-377092; Santa Cruz), phospho-PDHA (ab177461; Abcam), LDHA (ab84716; Abcam), PDK1 (ADI-KAP-PK112; Enzo), PDK3 (ab182574, Abcam), and GAPDH (sc-32233; Santa Cruz). HRP-conjugated anti-rabbit IgG or anti-rat IgG (all from Invitrogen) were used as secondary antibodies. The specific bands were developed using a western blot detection kit (Bio-Rad, Hercules, CA, United States) using the ImageQuant LAS 4000 chemiluminescence imaging system (GE Healthcare, Munich, Germany).
8
Immunofluorescence Analysis of Bax and Bcl-2
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To determine Bax and Bcl-2 expression pro les, an immuno uorescence staining was used as previously described (Onder et al. 2022 (link)). Cells were incubated on coverslips in a 24-well plate. After cells were treated with HES for 24 hours, they were xed in 10% formaldehyde. Then, the procedure of immuno uorescence staining was applied. Cells were treated with Bax (1:100, primary antibody, Novus Biologicals, Littleton, USA) and then Bcl-2 (1:30, primary antibody, Thermo, Rockford, USA). For each group, 400x images were obtained from ten separate microscopic elds using an Olympus BX51 uorescence microscope (Olympus, Tokyo, Japan). The intensity of Bax and Bcl-2 primary antibodies' immunoreactivity was measured using Image J software (Bethesda, USA).
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