Download Supplementary data Materials and methods 1.1. Plasmids pDEST27

Supplementary data
1. Materials and methods
1.1. Plasmids
pDEST27 and pDEST26 are mammalian expression vectors which encode N-terminal
GST-tagged and His-tagged fusion proteins, respectively. pDEST17 is bacterial expression
vector encoding N-terminal His-tagged fusion proteins. The pDEST27-LKB1 T336A plasmid
was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, USA).
AMPKα1 (1-312) was amplified from HeLa RNA using a PrimeScript™ One Step RT-PCR
Kit (Takara, Japan) followed by subcloning into the EcoR I/Xho I sites of the pGEX
(GST-tagged) bacterial expression vector. Wild type (WT) LKB1 and LKB1 T336A were
amplified from pDEST27-LKB1 WT or T336A and subcloned into the EcoR I/Bgl II sites of
pEGFP (a gift from Professor Mian Wu). DsRed-tagged 14-3-3 ζ was generated by
subcloning 14-3-3 ζ amplified from pDEST17-14-3-3 ζ into the EcoR I/Bgl II sites of
pDsRed.
1.2. Cell culture, transfection and lysis
COS7 cells were obtained from The Cell Bank of Type Culture Collection of Chinese
Academy of Sciences. Hela and HEK-293 cells were kindly provided by Professor Qinhua
Shi. Cells were maintained at 37 °C in Dulbecco’s modified Eagle’s medium (Invitrogen,
USA) containing 10% fetal bovine serum (HyClone, USA) and antibiotics (Sigma, USA).
Transient transfections were performed using Lipofectamine 2000 (Invitrogen, USA)
according to the manufacturer’s instructions. Cells were lysed in 1% NP-40 lysis buffer
containing 50 mM Tris-HCl pH 7.5, 1% NP-40, 1 mM sodium orthovanadate, 50 mM sodium
fluoride, 5 mM sodium pyrophosphate and complete Mini protease inhibitor cocktail tablets
(Roche, USA) and centrifuged at 4 °C for 5 min at 10,000 g .The supernatants were collected
and stored at -80 °C.
1.3. Protein expression and purification
The pDEST15-14-3-3 (ε, η, γ, τ, ζ, σ, ζ and K49E) and pGEX- AMPKα1 (1-312) plasmids
were transformed into BL21 E.coli. The cultures were grown at 37 °C to OD600= 0.6 followed
by addition of 500 μM Isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein
expression. The bacteria were further cultured for 5 h at 37 °C for the His-14-3-3 isoforms
and for 12 h at 20 °C for GST- AMPKα1. The bacteria were then harvested by centrifugation
for 20 min at 4,000g. Bacteria expressing the His-14-3-3 isoforms were resuspended in
ice-cold binding buffer (40 mM Imidazole, 4 M NaCl, 160 mM Tris-HCl pH 7.9), followed
by lysis with sonication (400 W). Cell lysate was centrifuged at 20,000g for 20 min. The
supernatant was collected, loaded onto a Ni2+ charged HiTrap IMAC HP column (GE
Healthcare, UK) and eluted with buffer containing 4 M Imidazole, 2 M NaCl and 80 mM
Tris-HCl pH 7.9. After desalination by dialysis, the purified His-14-3-3 proteins were stored
at -80 °C. Cells expressing GST- AMPKα1 were lysed in ice-cold lysis buffer followed by
sonication (400 W) and centrifugation at 20,000g for 20 min. The cleared cell lysate was
loaded onto a glutathione Sepharose column (GE Healthcare, UK). GST- AMPKα1 was
purified by affinity chromatography and removed from the resin by incubation with
PreScission protease (GE Healthcare, UK) in order to cleave the N-terminal GST tag.
Forty hours after transfection with pDEST27-LKB1 WT and pDEST27-LKB1 T336A,
COS7 cells were harvested by centrifugation for 5 min at 300g and lysed in ice-cold lysis
buffer. GST-LKB1 WT and GST-LKB1 T336A were purified by affinity chromatography with
glutathione Sepharose beads and eluted with buffer containing 10 mM reduced glutathione
and 50 mM Tris-HCl pH 8.0.
1.4. Antibodies
An antibody that specifically recognizes LKB1 that is phosphorylated at Thr336 was
generated by immunizing rabbits with a phospho-peptide containing the sequence
RWRSMpTVVPY (corresponding to residues 331-340 of human LKB1) conjugated to
keyhole limpet hemocyanine (KLH), where pT represents the phosphorylated Thr residue.
Serum from immunized rabbits was purified on an affinity column using rProtein A Sepharose
(GE Healthcare, UK) covalently coupled to the corresponding phosphorylated peptide.
Horseradish peroxidase (HRP) conjugated goat anti-mouse and goat anti-rabbit antibodies
(Cell Signaling, USA) were used as secondary antibodies for immunoblotting. Other
antibodies used in this study were obtained from commercial sources, including an antibody
recognizing the GST epitope (Proteintech group, USA), a His epitope monoclonal antibody
(Abmart, China), a LKB1 monoclonal antibody (Santa Cruz, USA), a pan 14-3-3 antibody
(Santa Cruz, USA), AMPKα1 (Epitomics, USA) and AMPKα1 phospho-Thr172 (Cell
Signaling, USA) antibodies and antibodies against S6K and S6K phospho-Thr389 (Bioworld,
USA).
1.5. Immunoprecipitation and immunoblotting
Endogenous LKB1 was immunoprecipitated using a monoclonal antibody specific for
LKB1 that was cross-linked to immobilized protein G using the Seize X Protein G
Immunoprecipitation Kit (Pierce, USA) according to the manufacturer’s instructions. The
resulting immunocomplexes were separated by SDS-PAGE and transferred to PVDF
membranes (Millipore, USA). The membranes were blocked with milk and probed with the
indicated primary antibodies and secondary antibodies conjugated to HRP. After incubation
with ECL (BestBio, China), the protein blots were visualized by exposure to Kodak X-OMAT
BT film.
1.6. LKB1 Kinase Assay
GST-LKB1 WT and GST-LKB1 T336A were ectopically expressed in COS7 cells.
GST-bound proteins were pulled down by Glutathione affinity chromatography and incubated
in reaction buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 250 μM cold ATP, 1m
M DTT, 10 mCi/ml [γ-32P]ATP) with purified AMPKα1 in the presence or absence of
His-14-3-3 ζ. After incubation for 30 min at 30 °C, the reaction was stopped by addition of
2×SDS sample buffer (0.5 M Tris-HCl pH 6.8, 4.4% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v)
2-mercaptoethanol, and 0.02% (w/v) bromophenol blue). The samples were then analyzed by
SDS-PAGE. After electrophoresis, the gel was dried at 80 °C and was exposed in an imager
cassette for 45 min with Kodak X-OMAT BT film.
1.7. Flow Cytometry
Forty hours after transfection, Hela cells were harvested and fixed in 70% ethanol
overnight at 20 °C. Fixed cells were washed three times with ice-cold PBS containing 0.2%
BSA, treated with RNase (1 mg/ml, Sigma-Aldrich) for 30 min at 37 °C. Nuclei were stained
with propidium iodide (30 mg/ml, Sigma) for 30 min in the dark. The cell cycle distribution
of Hela cells was determined by flow cytometric analysis (Becton Dickinson FACScan
system).
1.8. Subcellular localization of LKB1 and 14-3-3
Hela cells were transfected with EGFP-LKB1 WT, EGFP-LKB1 T336A, Flag-STRADα
and DsRed-14-3-3 ζ. Subcellular localization of the ectopically expressed proteins was
visualized by confocal microscopy (Olympus, FV1000).
2. Supplemental figures
Fig. S1 (a) LKB1 in COS7 has stronger affinity to STRAD and MO25 than that in
HEK-293. GST-LKB1 were expressed respectively in COS7 and HEK-293 cells and isolated
by GST affinity chromatography. Protein complexes respectively were detected by western
blot with GST, STRADα/β and MO25 α/β antibodies. (b) The mutation of Thr336 to Ala
does not change the association of LKB1 with STRAD and MO25. GST-LKB1 WT and
GST-LKB1 T336A were expressed in COS7 cells, and isolated by GST affinity
chromatography. Protein complexes respectively were detected by western blot with GST,
STRADα and MO25α antibodies.
Fig. S2 (a) Phosphorylation of Thr336 of GST-LKB1 T336A mutant could not be
detected by immunoblot with an antibody specific for phosphorylated Thr336 of LKB1.
GST-LKB1 WT and GST-LKB1 T336A were expressed in COS7 cells, and isolated by GST
affinity chromatography. Protein complexes were detected by western blot with GST and
LKB1 phospho-Thr336 antibodies. (b) His-14-3-3 ζ K49 abolishes the reaction between
14-3-3 and LKB1 in vitro. An equal quantity of each bacterially-expressed His-14-3-3 ζ and
His-14-3-3 ζ K49E were incubated with GST-LKB1 WT purified from COS7 cells.
GST-LKB1 WT was isolated by GST affinity chromatography. Protein complexes were
detected by western blot using GST and His antibodies.