Ac k2 100

Cells were washed twice with PBS and lysed in RIPA buffer. Protein concentrations were quantified using the Bradford reagent (Bio-Rad) according to the manufacturer's instructions. Samples with equal amounts of protein were separated by 4%–15% SDS-PAGE, then transferred to an Immobilon transfer membrane (Millipore) and immunoblotted with specific antibodies against E-cadherin, PCK2, PKM2, and Acetylated-Lysine (Ac-K²-100, #9814) from Cell Signaling, and GAPDH from Thermo Scientific. All of the immunoblots were visualized by enhanced chemiluminescene (Bio-Rad).
Immunofluoresence staining was performed to detect PCK2 expression and protein acetylation. Cells were plated in a 24-well multiwell glass-bottomed culture plate (MatTek), fixed in 4% formaldehyde in PBS for 15 min at room temperature, and then washed three times with PBS. After being blocked with PBS containing 5% goat serum and 0.3% Triton X-100 at room temperature for 1 h, the cells were incubated overnight with anti-PCK2 primary antibody (1:100, Cell Signaling) or anti- acetylated-Lysine (1:200, Cell Signaling #9441) at 4°C. The secondary antibody, Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes) was used at a 1:1000 dilution. Fluorescence was monitored by an inverted confocal laser microscope (Carl Zeiss).

Immunoprecipitation (IP) and western blotting (WB) were conducted as previously described (Cho et al. 2006 (link)). Briefly, transfected cells were lysed in cold lysis buffer and incubated with 1:200 dilutions of appropriate antibodies overnight at 4°C. The lysates were further incubated with 25 µL of protein A/G Plus agarose beads for 4 h (Santa Cruz Biotechnology). After incubation, IP complexes were washed and resuspended in loading buffer. Boiled samples were analyzed using 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with the following antibodies: GSK3α/β (sc-7291), poly (ADP-ribose) polymerase-1 (PARP-1; sc-8007), green fluorescent protein (GFP; sc-8334), and β-actin (sc-4778) (all from Santa Cruz Biotechnology); pS9-GSK3β (#9336) and Ac-K2-100 (#9814) (Cell Signaling Technology); Flag M2 (F3165; Sigma-Aldrich); and Myc (05-724; Millipore). The protein bands were developed using an enhanced chemiluminescence system (iNtRON Biotechnology).