Influence of glial biotin availability on coping up with ER stress
We hypothesize that biotin favors BCAA utilization and its deficiency accumulates BCAA to prolong autophagy inhibition and ER stress by chronic activation of mTORC1. Ganesan, D, Ramaian Santhaseela, A, Rajasekaran, S, Selvam, S, Jayavelu, T. Astroglial biotin deprivation under endoplasmic reticulum stress uncouples BCAA‐mTORC1 role in lipid synthesis to prolong autophagy inhibition in the aging brain. J. Neurochem. 2020; 00: 1– 14. https://doi.org/10.1111/jnc.14979
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Western Blotting: Protein concentrations of tissue were determined based on Lowry’s protocol (Lowry et al., 1951) and equal concentrations of proteins were resolved by Gradient gel (5-15%) SDS-PAGE. The proteins were transferred to nitrocellulose membrane and blocked with 5% skimmed milk powder and fatty acid free bovine serum albumin for total and phosphorylated proteins, respectively. Following this the membranes were probed with respective primary and secondary antibodies at appropriate dilutions and developed using SuperSignal West Femto Chemiluminescent Substrate. The signals were recorded by LI-COR Odyssey Fc imager and analyzed using Image studio software version 5.2. The signals were normalized against respective GAPDH signals and/or total proteins stained with ponceau S.