Journal: Cell & bioscience
Article Title: NUAK1 coordinates growth factor-dependent activation of mTORC2 and Akt signaling.
doi: 10.1186/s13578-023-01185-2
Figure Lengend Snippet: Fig. 1 NUAK1 interacts with mTOR and Rictor but not with Raptor upon EGF stimulation. A Table shows total peptide count (P), the distributed spectra count (dS), and distributed normalized spectral abundance (dNSAF), observed for each identified protein in murine FLAG-NUAK1 WT and FLAG-NUAK1 KR44/71AA purifications (n = 3). NE, nuclear extract; CE, cytoplasmic extract. B Immunoblot (IB) of the Immunoprecipitation (IP) of human FLAG-NUAK1 WT and CoIP of endogenous mTOR, MYPT1 and exogenous Myc-Raptor in HEK293T cells. C IB of the IP of FLAG-NUAK1 WT and CoIP of endogenous mTOR, MYPT1 and exogenous Myc-Rictor in HEK293T cells. D IB of the IP of FLAG-NUAK1 WT and CoIP of endogenous mTOR, Rictor, Raptor and MYPT1 in MDA-MB-231 cells. E, F IB of the IP of FLAG-NUAK1 WT and CoIP of endogenous mTOR, Rictor, Raptor and MYPT1 from MDA-MB-231 (E) and U87 (F) cells serum-starved overnight before stimulation with EGF by 10 min. G IB of the IP of endogenous Rictor and CoIP of endogenous mTOR, and NUAK1 in MDA-MB-231 cells serum-starved overnight before stimulation with EGF by 10 min. H Proximity ligation assay (PLA) in MDA-MB-231 cells expressing HA-tagged NUAK1, FLAG-tagged NUAK1 or Empty vector (EV) (used as a negative control). Cells were serum-starved overnight and stimulated with EGF by 10 min (n = 3). Red dots indicate proximity of HA-NUAK1 with MYPT1 (Positive control), HA-NUAK1 with Rictor or FLAG-NUAK1 with mTOR. DAPI was used as a nuclear counterstain
Article Snippet: To generate pCW57 FLAG-hNUAK1 WT, hNUAK1 WT was amplified from pCMV FLAG-hNUAK1 and subcloned into pCW57-MCS1-2 A-MCS2 using NheI and AgeI restriction sites. pCMV FLAG-hNUAK1 K84A was generated by subcloning of FLAG-hNUAK1 K84A from pBABE FLAG-hNUAK1 K84A using EcoRI restriction sites. pBABE FLAG-hNUAK1 K84A was previously generated by site direct mutagenesis using the following primers: hNUAK1_K84A_F: GGC CGA GTG GTT GCT ATA GCC TCC ATT CGT AAGG, hNUAK1_K84A_R: CCT TAC GAA TGG AGG CTA TAG CAA CCA CTC GGCC. pCMV FLAG-NUAK1 K84A mutation was confirmed by sequencing (Additional file 1: Fig. S6). pCW57MCS1-2 A-MCS2 (#71782), FLAG FOXO3a (#8360), pCMV dR8.2 (#8360), pCMV VSVG (#8454), pLKO scramble (#1864), pLKO shRictor (#1853), Lamp1-YFP (#2532), Lamp1-RFP (#1817), EGFR-GFP (#32751), pRK5 Myc Rictor (#11367), pRK5 Myc Raptor (#1859), and mRFP-Rab5 (#14437) from Addgene. pCMV6 Myr-Akt1HA was kindly provided by Dr. Philip Tsichlis, The Ohio State University, Rab7-GFP by Dr. Julio Tapia, Universidad de Chile, Chile, and pINDUCER10 shNUAK1 #1 and shNUAK1 #2 by Drs. Giacomo Cossa and Martin Eilers, University of Würzburg, Germany.
Techniques: Western Blot, Immunoprecipitation, Proximity Ligation Assay, Expressing, Plasmid Preparation, Negative Control, Positive Control
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