For sample preparation, when the cell culture reaches to 80% confluence, the cells were collected and lysed for 40 min on ice. Extracted protein samples were denatured in 5× loading buffer containing SDS in boiling water for 10 min. Five percent nonfat dry milk for detection of conventional proteins or 5% bovine serum albumin for detection of phosphorylated proteins dissolved in tris-buffered saline containing 0.1% Tween 20 was used for blocking and antibody dilution. The following antibodies and dilutions were used: MSN (1:2000, ab52490, Abcam), actin (1:1000, HC201, TransGen), FLAG (1:2000, F7425, Sigma-Aldrich), phosphorylated MSN (1:1000, ab177943, Abcam), lamin (1:1000, ab133256, Abcam), tubulin (1:2000, HC101, TransGen), NONO (1:1000, 11058-1-AP, Proteintech), pCREB [1:1000, 87G3, Cell Signaling Technology (CST)], CREB (1:1000, 48H2, CST), pPKCζ (1:1000, 9378S, CST), PKCζ (1:500, sc-17781, Santa Cruz Biotechnology), pPKA (1:1000, 4781, CST), PKA (1:1000, 4782, CST), goat anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP) (1:5000, HS201-01, TransGen), and goat anti-rabbit IgG-HRP (1:5000, HS101-01, TransGen). Western HRP Substrate (WBKLS0500, Millipore) was used to detect HRP-conjugated secondary antibodies. Software (ImageJ) was used to quantify the relative expression of proteins, which was presented as the ratio of test protein integrated density to internal control integrated density. All uncropped images from Western blots displayed in the study are presented in supplementary data (fig. S10).

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

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.