Total protein was extracted from cells or tissues using RIPA buffer (Beyotime) and quantified with an Easy II Protein Quantitative Kit (TransGen Biotech). The protein samples were subjected to SDS‐PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C. Then, HRP‐conjugated AffiniPure Goat Anti‐rabbit/mouse IgG secondary antibody (1:5000, Boster Biological Technology) was incubated for 1 hour. The EasySee® Western Blot Kit (TransGen Biotech) was used to detect the protein bands.
Bs 0127r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-0127R is a laboratory equipment product manufactured by Bioss Antibodies. It is a multi-functional device designed for various applications in scientific research and analysis. The core function of this product is to aid in the processing and handling of samples, materials, and solutions commonly used in laboratory settings. However, a more detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Easy 2 protein quantitative kit, by Transgene (1 mentions)
Ba3257, by Boster Bio (1 mentions)
Ma1 2021, by Thermo Fisher Scientific (1 mentions)
Bs 0013r, by Bioss Antibodies (1 mentions)
Ba2913, by Boster Bio (1 mentions)
Easysee western blot kit, by Transgene (1 mentions)
A00066 1, by Boster Bio (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab8932, by Abcam (1 mentions)
Ab196204, by Abcam (1 mentions)
Ab227204, by Abcam (1 mentions)
β actin, by Media Cybernetics (1 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions)
Rabbit anti tlr4, by Cell Signaling Technology (1 mentions)
Bs 3332r, by Bioss Antibodies (1 mentions)
Hairpin ittm microrna qpcr quantitation kit, by GenePharma (1 mentions)
Bs 4563r, by Bioss Antibodies (1 mentions)
Gb111114, by Wuhan Servicebio Technology (1 mentions)
Rabbit anti phospho nf κb p65, by Cell Signaling Technology (1 mentions)
6 protocols using bs 0127r
1
Protein Extraction and Western Blot Analysis
2
Hepatocyte Proliferation and Apoptosis
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Proliferative hepatocyte nuclei were quantified via staining with anti-Ki67 (ab15580, Abcam, Cambridge, UK), while apoptosis was analyzed via protein expression of Bax (bs-0127R, Bioss Antibodies, Woburn, MA, USA) and Bcl-2 (bs-4563R) as well as TUNEL (Sigma-Aldrich Corporation, St. Louis, MO, USA). For TUNEL, paraffin-embedded sections were processed; DNase I-incubated and Tdt-free samples were used as positive and negative controls, respectively. After incubation for 1 h in the dark, samples were co-stained with DAPI and prepared for fluorescence microscopy using Fluormont-G® mounting media (South Biotechnology Associates, Inc., Birmingham, UK). DAPI and TUNEL images at 400× magnification were merged in order to evaluate apoptotic cells.
3
Molecular Analysis of Signaling Pathways
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qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-itTM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.
4
Immunostaining of Brain Tissue Sections
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For brain tissue paraffin sections, after 1 h of dewaxing, antigen retrieval was performed with sodium citrate solution at 90 °C for 15 min, followed by 45 min of blocking with 10% BSA for immunostaining at room temperature, overnight incubation with primary antibodies at 4 °C, and washing three times with PBS, then incubation with appropriate secondary antibodies for 1 h at room temperature. The primary antibodies include rabbit anti-NLRP3 (PAB38738, Bioswamp, 1:200), rabbit anti-MARK4 (AF0693, Affinity, 1:200), rabbit anti-GFAP (bs-0199R, Bioss, 1:200), rabbit anti-Iba1 (A19776, ABconal, 1:200), rabbit anti-BAX (bs-0127R, Bioss, 1:200). The secondary antibody used is donkey anti-rabbit Alexa Fluor568 (A10042, Invitrogen, 1:1000). Images were captured using a fluorescence microscope (Nikon, Tokyo, Japan), and image analysis was performed using Image J.
5
Protein Extraction and Western Blot Analysis
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Protein extraction was finished using RIPA buffer (Beyotime) and protein concentration determination was executed utilizing a BCA Protein Quantification Kit (Vazyme). 20 μg proteins were separated by 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) electrophoresis. Next, the proteins were blotted onto polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). Afterward, the membranes were blocked utilizing 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies against GAPDH (bs-2188R; 1:5,000; Bioss, Beijing, China), B-cell lymphoma-2 (Bcl-2; 1:2,000; bs-34012R; Bioss), BCL2-Associated X (Bax; 1:2,000; bs-0127R; Bioss), cleaved caspase-3 (bs-0081R; 1:2,000; Bioss), and PDE7B (bs-11576R; 1:2,000; Bioss) overnight at 4°C and secondary antibody (bs-0295M-HRP; 1:5,000; Bioss) for 1 h at room temperature, followed by ECL detection.
6
Western Blot Analysis of Cellular Signaling
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Cells treated as described earlier were washed three times with PBS and lysed in RIPA buffer with a 1% protease inhibitor. Cell lysates were centrifuged, and the protein concentration was measured using a BCA assay kit. Equal protein aliquots (10 μg) were fractionated by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 3% bovine serum albumin in TBST and incubated with primary antibodies of Bax (bs-0127R, Bioss, Beijing, China), p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), PCNA (bs-2006R, Bioss, Beijing, China), LC-3 (12741S, CST, Boston, United States), Atg-5 (10181-2-AP, Proteintech, Wuhan, China), Beclin-1 (bs-1353R, Bioss, Beijing, China), NF-κB (10745-1-AP, Proteintech, Wuhan, China), p-NF-κB (bs-0982R, Bioss, Beijing, China), STING (19851-1-AP, Proteintech, Wuhan, China), cGAS (ab252416, Abcam, Cambridge, United Kingdom), iNOS (ab15323, Abcam, Cambridge, United Kingdom), GBP5 (13220-1-AP, Proteintech, Wuhan, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), and GAPDH (PMK053C, BioPM, Wuhan, China) overnight at 4°C, and then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were developed using an ECL, and the films were exposed using a bio-imaging system (170-8265, Bio-Rad).
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