Cytoplasmic and nuclear proteins were extracted from cells using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Protein concentrations were determined using a BCA assay. Western blotting was performed as previously described with minor modifications (18). Proteins (50 µg) were separated via 10% SDS-PAGE and transferred to PVDF membranes (20 V; 100 mA) overnight. Following blocking with 5% skimmed milk at 37°C for 4 h, the membranes were incubated with primary antibodies (all 1:1,000) targeted against: CD40 (cat. no. sc-59047), ICAM-1 (cat. no. sc-8439), VCAM-1 (cat. no. sc-13160), iNOS (cat. no. sc-7271), NF-κB p65 (cat. no. sc-166748), p-IκB-α (cat. no. sc-8404), IκB-α (cat. no. sc-1643) and IKK-β (cat. no. ab32135) at 4°C for 24 h. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (cat. no. sc-2005; 1:800) at 25°C for 12 h. Protein bands were visualized using an ECL Plus kit and a Gel Imaging System (Thermo Fisher Scientific, Inc.). Protein expression was semi-quantified using Quantity One software (version 4.0; Bio-Rad Laboratories, Inc.). Histone and β-actin were used as the loading controls for nuclear and cytoplasmic proteins, respectively.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.