Antibody Sourcing and Western Blot Protocol

Reagents were obtained from the following sources. Monoclonal antibody phospho-T389 S6K1, rabbit antibody S6K, and rabbit antibody ATF4, which could react with antigens from rat, were obtained from Cell Signaling Technology. Rabbit antibody LC3 was obtained from MBL, rabbit antibody β-actin was obtained from Sigma-Aldrich, and rabbit antibody SLC7A5 was obtained from Cosmo Bio Company and could react with antigens from rat. 2-Aminobicyclo-(2, 2, 1)-heptane-2-carboxyl acid was obtained from Sigma-Aldrich. EvaGreen was obtained from Biotium and Trizol from Invitrogen. RNeasy Mini kit was purchased from QIAGEN, RevertAid First Strand cDNA Synthesis kit from Fermentas, BCA Protein Assay kit from Thermo Fisher Scientific, and Fluoromount-G from SouthernBiotech. Western blots were performed as described previously (Yu et al., 2010 (link)).

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Chen R., Zou Y., Mao D., Sun D., Gao G., Shi J., Liu X., Zhu C., Yang M., Ye W., Hao Q., Li R, & Yu L. (2014). The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation. The Journal of Cell Biology, 206(2), 173-182.

Publication 2014

phospho-T389 S6K1 β-actin EvaGreen Trizol RNeasy Mini kit RevertAid First Strand cDNA Synthesis kit BCA Protein Assay kit Fluoromount-G

Actin Antibody Antigens Assay Atf4 Carboxyl acid Cdna Heptane Monoclonal antibody Protein Rabbit Slc7a5 Synthesis Trizol Western blots

Corresponding Organization : Center for Life Sciences

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Variable analysis

independent variables

2-Aminobicyclo-(2, 2, 1)-heptane-2-carboxyl acid

dependent variables

Protein expression levels of phospho-T389 S6K1, S6K, ATF4, LC3, β-actin, and SLC7A5

control variables

Monoclonal antibody phospho-T389 S6K1, rabbit antibody S6K, and rabbit antibody ATF4, which could react with antigens from rat
Rabbit antibody LC3
Rabbit antibody β-actin
Rabbit antibody SLC7A5, which could react with antigens from rat
EvaGreen
Trizol
RNeasy Mini kit
RevertAid First Strand cDNA Synthesis kit
BCA Protein Assay kit
Fluoromount-G

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