Adherent fibroblasts were washed with ice-cold phosphate-buffered saline and then lysed using PhosphoSafe extraction reagent (Merck Millipore) supplemented with protease inhibitor (Merck Millipore). Equal protein quantities of lysate were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose, and protein levels were assessed by Western blotting with the following antibodies: p70S6K [Cell Signaling Technology (CST) #9202], phospho-p70S6KThr389 (CST #9234), 4E-BP1 (CST #9644), phospho-4E-BP1Ser65 (CST #9451), SMAD3 (CST #9523), Rictor (CST #2114), Raptor (CST #2280), GLUT1 (Abcam #EPR3915), PHGDH (CST #13428), PSAT (Thermo Fisher Scientific #PA522124), PSPH (Insight Biotechnology #GTX109163-S), SHMT2 (CST #12762), ATF4 (CST #11815), α-tubulin (CST #9099), histone H4 (Abcam #16483), phospho-eIF2α (Ser51) (CST #3597), and eIF2α (#9722). The dilutions of primary and secondary antibodies were according to the manufacturer’s instructions. Tunicamycin (2 μg/ml; MP Biomedicals) was used as a positive control for immunostaining of p-eIF2α. Protein band intensity was measured by densitometry in ImageQuant TL v8.1 (GE Healthcare) after electrochemiluminescence. All densitometries are presented relative to α-tubulin unless otherwise stated.

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.