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).
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).