The ICB complex was homogenized and solubilized in a cell lysis solution containing 1% protease inhibitors [19 (link)].The total protein concentration was determined by the BCA protein assay kit. In addition, 60 μg of protein lysate was resolved in 10% SDS–PAGE and then transferred onto nitrocellulose membranes (Millipore, Billerica, MA). The blots of non-phosphorylation and phosphorylation were blocked with 5% non-fat dry milk or 5% BSA, respectively, and probed overnight at 4 °C with primary antibodies specific to NF-κB p65 (1:1200; ABCAM, Cambridge, UK), phosphorylated p38 MAPK(1:1000; ABCAM, Cambridge, UK), and p38 MAPK(1:1000; ABCAM, Cambridge, UK). The blots were washed with Tris-buffered saline with Tween-20 and incubated with an HRP-conjugated secondary antibody for 1 h at 37 °C. Blots were developed using enhanced chemiluminescence (Amersham, Piscataway, NJ). Bands were analyzed using Image J software (Version1.43, Broken Symmetry Software, Bethesda, MD). An NF-κB p65 analysis was normalized against a housekeeping protein β-actin and a phosphorylated p38 MAPK analysis was normalized against p38 MAPK. The measurements were repeated 3X in each experiment (n = 4 per group).
Primary antibodies specific
Manufactured by Abcam
Sourced in United Kingdom
Primary antibodies are a class of antibodies that specifically bind to target antigens, enabling the detection and study of these target proteins in various applications such as immunoassays, Western blotting, and immunohistochemistry. These antibodies are a crucial tool for researchers in the fields of biology, biochemistry, and medicine.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
P38 mapk, by Abcam (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Phosphorylated p38 mapk, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
3 protocols using primary antibodies specific
1
Western Blot Analysis of NF-κB p65 and p38 MAPK
2
Immunophenotyping Differentiated ADSCs
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Immunocytochemistry analysis was performed as previously described (Kang et al., 2019 (link)). To identify the phenotypic changes in differentiated ADSCs, the expression levels of SC markers were recognized by immunophenotype using primary antibodies specific for S100ß (1:100, Abcam), NGFR (1:100, Cell Signaling Technology), MPZ (1:100, Abcam) and GFAP (1:300, Cell Signaling Technology). Uninduced ADSCs were used as a negative control. The images were taken by fluorescence microscopy.
3
Protein Extraction and Western Blot Analysis
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The proteins were extracted from the hippocampus of mice and HT22 cells using RIPA lysis buffer (Beyotime, Shanghai, China). The concentration of protein was determined using a BCA kit (Beyotime, Shanghai, China). The protein was separated by SDS- PAGE, followed by transfer onto PVDF membranes. Subsequently, the membranes were incubated with primary antibodies specific for caspase-3 or β-actin (1:1000, Abcam). Then the membranes were incubated with secondary antibodies (1:10,000, Abcam), and bands were visualized with an ECL Chemiluminescent Substrate Reagent kit (Beyotime, Shanghai, China). Image J software was used to analyze the bands, and the expression ratios were normalized to β-actin.
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