Gst beads

For the GST pull-down assay, GST-fusion proteins were expressed and purified according to the manufacturers’ instructions (Amersham Pharmacia and Qiagen). Cell lysates expressing target proteins were incubated with GST-fusion proteins bound to GST beads (Amersham Pharmacia), and the pull-down proteins were examined. For the coimmunoprecipitation assay, cells were harvested and lysed in lysis buffer on ice for 30 min. After centrifugation at 4 °C at 13,800 × g for 15 min, antibodies were added to the supernatant with rolling at 4 °C overnight. Protein G or A agarose (Santa Cruz) was then added to the samples, and the samples were rolled at 4 °C for 2 h. After the beads were washed three times with lysis buffer, the pellets were dissolved into 2 × SDS loading buffer after centrifugation and boiled at 100 °C for 10 min. Proteins were analyzed by immunoblotting with the indicated antibodies.

GST-LATS1-142-704 was obtained by PCR amplification from the vector p2xFlag CMV2 LATS1 (a gift from Marius Sudol) and cloned into pGEX4T3 with adapters for BamHI and NotI. GST-FAT1-ICD was generated by subcloning FAT1-ICD from pCEFL CD4-FAT1-ICD into the BamHI and NotI sites of pGEX 4T3. Both GST fusion proteins, and GST as a control, were expressed in BL21 E.coli transfected with each pGEX4T3 construct and coupled to GST-Sheparose beads. To incubate the GST beads with mammalian cell extracts, HEK293 cells were cultured in 15 cm plates and, upon reaching confluency, they were quickly washed twice in ice-cold PBS and then lysed in pulldown buffer (1 mL of 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 0.3% (w/v) CHAPS, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 500 μM sodium orthovanadate, 10 μg/mL aprotinin, and 10 μg/mL leupeptin and 1 mM PMSF). The pulldown was performed by adding 20 µL of beads (or 10 µg of protein) to 1 mL of cell lysate and incubated at 4 °C for 2 h with their corresponding GST beads (Amersham, Piscataway, NJ). Beads were then washed three times with pulldown buffer, resuspended in Laemmli sample buffer and resolved by SDS-PAGE.

pGEX-6P2 containing wild and nsSVPs of PKM2 were transformed in E. Coli strain BL21-DE3 by the heat shock method. Single colony was inoculated in LB medium overnight at 37°C under shaking conditions, followed by 1% inoculation of overnight grown culture in 200ml fresh LB. The culture was grown at 37°C for 2 hrs (till mid log phase) and induced with 1mM IPTG at 18°C for 12 hrs when culture was harvested by centrifugation and pellet stored at -80°C. For protein purification, the cell pellet was lysed in sonication buffer containing 50mM Tris-HCl (pH = 8), 500mM NaCl, 10% glycerol, 1mM PMSF and 1mM 2-mercaptoehtanol. The culture was lysed by an intermittent pulse of sonication and finally centrifuged at high speed to get a clear lysate. The supernatant was incubated with 400ul of GST beads (Amersham) for 4 hrs and beads washed 3–5 times, using 10 ml of sonication buffer. In order to obtain the GST tag free PKM2 protein, a fraction of washed beads was incubated with 80-U of Pre-Scission protease (GE Life sciences) at 5°C for 4 hours in prescribed conditions. Beads were then pelleted and the supernatant collected at 4°C. Protein quality was checked on running a fraction on 12% SDS PAGE and quantified using BCA protein estimation method (Pierce thermo scientific).