Fig. 3.

Biotin uncouples mTORC1-dependent protein synthesis. Effect of biotin on upstream and downstream candidates of mTORC1 signaling were determined using immunoblot. Total cell lysates for immunoblot were prepared on the sixth day of differentiation using cell lysis buffer containing protease and phosphatase inhibitors cocktail. Equal amount of protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The Ponceau S-stained membranes were used to normalize protein loading. The values were expressed as average fold changes of three independent experiments. The ratio of phosphorylated versus total levels of respective proteins indicates their phosphorylation status