HUVECs were lysed with RIPA buffer (Beyotime, Shanghai, China), and total protein was obtained. The protein concentration was measured by using the BCA protein assay kit (Tiangen). After processing with loading buffer, proteins (50 µg) were subjected to SDS/PAGE (10–12% gels), and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The blot was blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies used were listed as following: RPS4Y1 (1:1000; RayBiotech, Norcross, GA, USA), NOX-4 (1:1000; Abcam), IL-1β (1:1000; Abcam), IL-6 (1:1000; Abcam), TNF-α (1:1000; Abcam), p38 (phospho Y182, 1:1000; Abcam), ERK1/2 (1:1000; Abcam), ERK1/2 (phospho T202/T185, 1:1000; Abcam), Jnk1/2 (1:1000; Abcam), Jnk1/2 (phosphor T183/Y185), β-actin (1:1000; Abcam), IL-8 (1:1000; Biorbyt, San Francisco, CA, USA), and p38 (1:500; Biorbyt). After washing, blots were incubated with appropriate HRP-conjugated secondary antibody (1:1000; Abcam) for 2 h. The protein bands were visualized and revealed by chemiluminescence using an ECL detection kit (Millipore).
Jnk1 2
Manufactured by Abcam
Sourced in United Kingdom
JNK1/2 is a protein kinase that belongs to the MAP kinase family. It is involved in the regulation of various cellular processes, including cell growth, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Erk1 2, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Tnf α, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Abcam (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Mouse anti mkp 1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Goat anti iba1 antibody, by Abcam (1 mentions)
3 protocols using jnk1 2
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Signaling Pathways
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The cell in 96-well plates were treated as described above and then stimulated with LPS (200 ng/mL) for 24 h. The cells were harvested and lysed in an extraction lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors. The protein concentration was determined using a BCA protein assay kit (Thermo Scientific, 23227). The whole cell lysates were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Each membrane was incubated with Tris-buffered saline (pH 7.6, containing 0.05% Tween-20 and 5% non-fat milk). The nitrocellulose membrane was incubated with the primary antibody against p-JNK1/2, JNK1/2, p-p38, p38, p-ERK1/2, ERK1/2, IκBa, NF-κB p65, COX-2, iNOS or β-Actin (Abcam, Cambridge, UK). Immunoreactive bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies, and visualised using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA).
3
Western Blot Analysis of Signaling Pathways
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After being lysed and centrifugated, the supernatant was determined by BCA kit. After being normalized and boiled with loading buffer, the proteins were separated and transformed into PVDF membrane (Millipore, Burlington, Massachusetts). After being treated with 5% bovine serum albumin for 1 hour, membranes were incubated with primary antibodies in 1% bovine serum albumin. In this study, rabbit anti-p-p38MAPK, p38MAPK, p-ERK1/2, ERK1/2, RXRα (Cell Signaling Technology, Danvers, Massachusetts; 1:1,000), rabbit anti-p-JNK1/2 (Abcam, Cambridge, UK; 1:2,000), JNK1/2 (Abcam; 1:5,000), mouse anti-MKP-1 (Santa Cruz Technology, Santa Cruz, California; 1:500), goat anti-iba-1 antibody (Abcam; 1:1,000), rabbit GAPDH and anti-β-actin (ABclonal Technology, Woburn, Massachusetts; 1:5,000) were used. After being washed, membranes were treated with horseradish peroxidase-linked antirabbit or mouse secondary antibodies (Cell Signaling Technology, 1:1,000) for 1 hour. Signals were visualized by with horseradish peroxidase substrate (Millipore; Luminata Forte).
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