KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
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| Antibodies | ||
|
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| phospho-AKT2(T308) | Cell Signaling Technology | Cat# 13038; RRID: AB_2629447 |
| AKT2 | Cell Signaling Technology | Cat# 3063; RRID: AB_2225186 |
| phospho-RSK2(S227) | Cell Signaling Technology | Cat# 3556; RRID: AB_2181465 |
| RSK2 | Cell Signaling Technology | Cat# 5528; RRID: AB_10860075 |
| phospho-AS160(T642) | Cell Signaling Technology | Cat# 8881; RRID: AB_2651042 |
| AS160 | Cell Signaling Technology | Cat# 2670; RRID: AB_2199375 |
| phospho-GSK3β(S9) | Cell Signaling Technology | Cat# 5558; RRID: AB_10013750 |
| GSK3β | Cell Signaling Technology | Cat# 12456; RRID: AB_2636978 |
| phospho-TSC2(S939) | Cell Signaling Technology | Cat# 3615; RRID: AB_2207796 |
| TSC2 | Cell Signaling Technology | Cat# 3990; RRID: AB_2209986 |
| phospho-PRAS40(T246) | Cell Signaling Technology | Cat# 13175; RRID: AB_2798140 |
| PRAS40 | Cell Signaling Technology | Cat# 2691; RRID: AB_2225033 |
| PDK1 | Cell Signaling Technology | Cat# 5662; RRID: AB_10839264 |
| GLUT4 | Cell Signaling Technology | Cat# 2213; RRID: AB_823508 |
| Na-K-ATPase | Cell Signaling Technology | Cat# 3010; RRID: AB_2060983 |
| GAPDH | Cell Signaling Technology | Cat# 5174; RRID: AB_10622025 |
| phospho-IR(Y1150/1151) | Cell Signaling Technology | Cat# 3024; RRID: AB_331253 |
| IR | Cell Signaling Technology | Cat# 3020; RRID: AB_2249166 |
| phospho-IRS1(Y895) | Cell Signaling Technology | Cat# 3070; RRID: AB_2127863 |
| IRS1 | Cell Signaling Technology | Cat# 2390; RRID: AB_10692516 |
| phospho-AKT2(S473) | Cell Signaling Technology | Cat# 4060; RRID: AB_2224726 |
| CDK2 | Cell Signaling Technology | Cat# 2546; RRID: AB_2276129 |
| SIRT3 | Cell Signaling Technology | Cat# 5490; RRID: AB_10828246 |
| COX4 | Cell Signaling Technology | Cat# 4884; RRID: AB_2085427 |
| PGK1 | Abcam | Cat# ab38007; RRID: AB_2161220 |
| PI3K p110α | Cell Signaling Technology | Cat# 4255; RRID: AB_659888 |
| PI3K p85 | Cell Signaling Technology | Cat# 4292; RRID: AB_329869 |
| Flag | Sigma-Aldrich | Cat# F3165; RRID: AB_259529 |
| DHA | LSbio | Cat# LS-C714681; |
| EPA | LSbio | Cat# LS-C714683; |
| AKT1 | Abcam | Cat# ab89402; RRID: AB_2049150 |
| phospho-AKT1(S473) | Cell Signaling Technology | Cat# 9018; RRID: AB_2629283 |
| phospho-AKT1(T308) | Abcam | Cat# ab105731; RRID: AB_10900490 |
| phospho-S6K(T389) | Cell Signaling Technology | Cat# 9234; RRID: AB_2269803 |
| S6K | Cell Signaling Technology | Cat# 9202; RRID: AB_331676 |
| BiP | Cell Signaling Technology | Cat# 3177 RRID: AB_2119845 |
|
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| Chemicals, peptides, and recombinant proteins | ||
|
| ||
| DHA | Sigma-Aldrich | Cat# D8768 |
| Methyl-DHA | Sigma-Aldrich | Cat# D2659 |
| Ethyl-DHA | Sigma-Aldrich | Cat# D2410 |
| EPA | Sigma-Aldrich | Cat# E6627 |
| Methyl-EPA | Sigma-Aldrich | Cat# CRM47571 |
| Ethyl-EPA | Sigma-Aldrich | Cat# E0085000 |
| linoleic acid | Sigma-Aldrich | Cat# L8134 |
| palmitic acid | Sigma-Aldrich | Cat# P9767 |
| oleic acid | Sigma-Aldrich | Cat# O7501 |
| conjugated linoleic acid | Sigma-Aldrich | Cat# 16413 |
| linolenic acid | Sigma-Aldrich | Cat# L2378 |
| Insulin | Sigma-Aldrich | Cat# 1342106 |
| Tryptophan | Sigma-Aldrich | Cat# V900470 |
| methyl-tryptophan | Sigma-Aldrich | Cat# 447439 |
| Tryptamine | Sigma-Aldrich | Cat# 246557 |
| DTT | Sigma-Aldrich | Cat# D9760 |
| Wortmannin | Sigma-Aldrich | Cat# W1628 |
| Alanine | Sigma-Aldrich | Cat# 05130 |
| 5-hydroxytryptamine | J&K Scientific Ltd. | Cat# 153–98-0 |
| L-α-phosphoatidylserine | Avanti | Cat# 870336C |
| Peptides, see Figure S2C | GL Biochem (Shanghai) Ltd. | N/A |
| Penicillin-Streptomycin | Invitrogen | Cat# 15070063 |
|
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| Critical commercial assays | ||
|
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| Mouse PIP3 ELISA | Shanghai Enzyme-linked Biotechnology Co., LTD. | Cat# ml037511–1 |
| Glucose Uptake-GloTM Assay Kit | Promega | Cat# J1341 |
| AKT kinase activity assay kit | Abcam | Cat# ab139436 |
| PDK1 kinase activity assay kit | Promega | V9681 |
|
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| Deposited data | ||
|
| ||
| Raw phosphoproteomics data | N/A | Mendeley data: https://doi.org/10.17632/rc3djvsgn3.1 |
| Raw targeted proteomics data | N/A | Mendeley data: https://doi.org/10.17632/n4npj2j8rx.1 |
|
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| Experimental models: Cell lines | ||
|
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| Mouse: 3T3-L1 | Stem Cell Bank, Chinese Academy of Sciences | Cat# GNM25 |
| Cricetulus griseus: CHO-K1 | Stem Cell Bank, Chinese Academy of Sciences | Cat# GNHa 4 |
| Human: HepG2 | Stem Cell Bank, Chinese Academy of Sciences | Cat# SCSP-510 |
| Human: HEK293T | Stem Cell Bank, Chinese Academy of Sciences | Cat# GNHu17 |
|
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| Experimental Models: Organisms/strains | ||
|
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| Mouse: 129/C57BL6 | Shanghai SLAC Laboratory Animal Co.,Ltd | N/A |
| Mouse: 129/C57BL6, AKT2 KO | Shanghai SLAC Laboratory Animal Co., Ltd | N/A |
|
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| Recombinant DNA | ||
|
| ||
| Myc-GLUT4-mCherry | This work | N/A |
| AKT2-EGFP | This work | N/A |
| AKT2R25C-EGFP | This work | N/A |
| AKT2R25C-Flag | This work | N/A |
| PDK1-EGFP | This work | N/A |
| PDK1R474A-Flag | This work | N/A |
| PDK1W448/543L-EGFP | This work | N/A |
| PDK1w266/347L-EGFP | This work | N/A |
| PDK1-Flag | This work | N/A |
| PDK16W/L-Flag | This work | N/A |
| PDK1W448/543L-Flag | This work | N/A |
| PDK1W266/347L-Flag | This work | N/A |
| PDK1R474A-Flag | This work | N/A |
| AKT2-Flag | This work | N/A |
| AKT27W/L-Flag | This work | N/A |
| AKT2W414L-Flag | This work | N/A |
| AKT2W414L-EGFP | This work | N/A |
| AKT2W334L-Flag | This work | N/A |
| AKT2W334L-EGFP | This work | N/A |
| AKT1-Flag | This work | N/A |
| AKT1W413L-Flag | This work | N/A |
| PGK1-EGFP | This work | N/A |
| PGK1-Flag | This work | N/A |
| PGK1-PH-EGFP | This work | N/A |
| PGK1-PH-Flag | This work | N/A |
| PKM2-Flag | This work | N/A |
|
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| Software and algorithms | ||
|
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| Graphpad Prism | Graphpad Software | https://www.graphpad.com/scientific-software/prism/ |
| Image J | NIH | https://imagej.nih.gov/ij/ |
| MaxQuants | Max planck institute of biochemistry | https://www.maxquant.org |
| Perseus software | Max planck institute of biochemistry | https://maxquant.net/perseus/ |