Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (mouse) | 3T3-L1 | Dr Howard Green, Harvard Medical School | RRID:CVCL_0A20 | |
| Cell line (human) | HEK293E | American Type Culture Collection | RRID:CVCL_6974 | |
| Cell line (human) | HeLa | American Type Culture Collection | RRID:CVCL_0030 | |
| Cell line (human) | HCC1937 | American Type Culture Collection | RRID:CVCL_0290 | |
| Cell line (human) | MCF7 | Associate Prof. Jeff Holst, Centenary Institute | RRID:CVCL_0031 | |
| Antibody | pAkt T309 (rabbit polyclonal) | Cell Signalling Technology | Cat#9275 | WB (1:1000) |
| Antibody | pAkt T309 (rabbit monoclonal) | Cell Signalling Technology | Cat#13038 | WB (1:1000) |
| Antibody | pAkt S474 (mouse monoclonal) | Cell Signalling Technology | Cat#4051 | WB (1:1000) |
| Antibody | pAS160 T642 (rabbit polyclonal) | Cell Signalling Technology | Cat#4288 | WB (1:1000) |
| Antibody | pPRAS40 T246 (rabbit monoclonal) | Cell Signalling Technology | Cat#2997 | WB (1:1000) |
| Antibody | pGSK3ɑ/β S21/29 (rabbit monoclonal) | Cell Signalling Technology | Cat#9327 | WB (1:1000) |
| Antibody | pFOXO1 S256 (rabbit polyclonal) | Cell Signalling Technology | Cat#9461 | WB (1:1000) |
| Antibody | 14-3-3 (rabbit polyclonal) | Santa Cruz | Cat#sc-629 | WB (1:1000) |
| Antibody | pP70S6K T389 (rabbit polyclonal) | Cell Signalling Technology | Cat#9205 | WB (1:1000) |
| Antibody | Akt2 (rabbit monoclonal) | Cell Signalling Technology | Cat#3063 | WB (1:1000) |
| Antibody | Caveolin (mouse monoclonal) | Abcam | Cat#ab17052 | WB (1:1000) |
| Antibody | Pan Akt (rabbit monoclonal) | Cell Signalling Technology | Cat#4685 | WB (1:1000) |
| Antibody | PI3K p85 (rabbit monoclonal) | Cell Signalling Technology | Cat#4257 | WB (1:1000) IF (1:100) |
| Antibody | PI3K p110 (rabbit monoclonal) | Cell Signalling Technology | Cat#4249 | WB (1:1000) |
| Antibody | IRS1 (rabbit polyclonal) | Cell Signalling Technology | Cat#2382 | WB (1:1000) |
| Antibody | Na,K-ATPase (rabbit polyclonal) | Prof. Gus Lienhard | ||
| Antibody | IR (rabbit monoclonal) | Cell Signalling Technology | Cat#3025 | WB (1:1000) |
| Antibody | pTyrosine (rabbit monoclonal mix) | Cell Signalling Technology | Cat#8954 | WB (1:1000) |
| Antibody | ɑ-Tubulin (mouse monoclonal) | Sigma-Aldrich | Cat#T9026 | WB (1:1000) |
| Antibody | IRS1 (rabbit monoclonal) | Cell Signalling Technology | Cat#3407 | IP (2 µL per sample) |
| Antibody | IRS2 (rabbit polyclonal) | Cell Signalling Technology | Cat#3089 | IP (2 µL per sample) |
| Antibody | FLAG (mouse monoclonal) | Sigma-Aldrich | Cat#F1804 | IP (5 µg per sample) |
| Antibody | IgG control (rabbit) | Santa Cruz | Cat#sc-2027 | IP (control) |
| Antibody | Alexa Fluor 555 (goat polyclonal) | Thermo Fisher Scientific | Cat#A21428 | IF(1:200) |
| Peptide, recombinant protein | 3X FLAG Peptide | Sigma-Aldrich | Cat#F4799 | |
| Chemical compound, drug | Insulin | Sigma-Aldrich | Cat# I5500; CAS 11070-73-8 | |
| Chemical compound, drug | Insulin-like growth factor 1 (IGF1) | Miltenyi Biotec | Cat#130-093-886 | |
| Chemical compound, drug | Epidermal growth factor (EGF) | Miltenyi Biotec | Cat#130-097-749 | |
| Chemical compound, drug | GDC0068 | Selleck Chemicals | Cat#S2808; CAS 1001264-89-6 | |
| Chemical compound, drug | MK2206 | MedChemExpress | Cat#HY-10358; CAS 1032350-13-2 | |
| Chemical compound, drug | Wortmannin | Sigma-Aldrich | Cat#W1628; CAS 19545-26-7 | |
| Chemical compound, drug | Rapamycin | LC Laboratories | Cat#R-5000; CAS 53123-88-9 | |
| Chemical compound, drug | Rapalink | Prof. Kevan Shokat (Rodrik-Outmezguine et al., 2016) | ||
| Chemical compound, drug | GSK2334470 | Tocris | Cat# 4143; CAS 1227911-45-6 | |
| Commercial assay or kit | PIP3 ELISA | Echelon Biosciences | Cat#K-2500S | |
| Commercial assay or kit | PI(3,4)P2 ELISA | Echelon Biosciences | Cat#K-4500 | |
| Recombinant DNA reagent | TagRFP-T-Akt2 (plasmid) | Norris et al., 2017 | ||
| Recombinant DNA reagent | TagRFP-T-Akt2 W80A; W80A-T309A; W80A-S474A; W80A-T309A-S474A; W80A-K181A (plasmids) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | PDPK1-eGFP (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | PDPK1-TagRFP-T (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | PH-Gab2-GFP (plasmid) | Sergio Grinstein – Addgene | Plasmid #35147 | |
| Recombinant DNA reagent | PI3K p110* (plasmid) | Morris Birnbaum | ||
| Recombinant DNA reagent | pMIG FLAG-Akt2 W80A; W80A-T309A (plasmids) | Kearney et al., 2019 | ||
| Recombinant DNA reagent | IRS1-eGFP (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS1-eGFP S270A; S307A; S330A; T525A; S527A; S1101A; S270A-S307A-S330A-T525A-S527A-S1101A (6P); S270A-S527A; DelPH; DelPTB (plasmids) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS2-eGFP (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS2-eGFP S306A; S346A; S365A; S577A; S1149A; S306A-S346A-S365A-S577A-S1149A (5P); S306A-S577A (plasmids) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS2-FLAG (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Sequence-based reagent | DNA Primers | Sigma-Aldrich | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Software, algorithm | MaxQuant | Cox and Mann, 2008; Tyanova et al., 2016 | RRID:SCR_014485 | |
| Software, algorithm | Image Studio | LI-COR Biosciences | RRID:SCR_013715 | |
| Software, algorithm | Graphpad Prism | GraphPad Software Inc. | RRID:SCR_002798 | |
| Software, algorithm | Fiji ImageJ | Schindelin et al., 2012 | RRID:SCR_003070 | |
| Software, algorithm | Tableau Prep | Tableau Software | ||
| Software, algorithm | R | https://www.R-project.org/ | RRID:SCR_001905 | |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | IQM | IntiQuan |