Anti pkm2 primary antibody

PKM2 subcellular localization was detected by immunofluorescence. After 24 h of attachment in cell chamber slides (Millipore), cells were treated with OXA and fixed to coverslips in cold acetone for 10 min at room temperature. Blocking and permeabilization was done with PBS-T/ FBS 10%. Cells were incubated at room temperature with a rabbit polyclonal anti-PKM2 primary antibody (Cell Signaling; 1:100) for 1.5 h and subsequently, with secondary antibody anti-rabbit Alexa-568 (Invitrogen; 1:200). Nuclei were stained with DAPI gold-antifade reagent (Invitrogen). Coverslips were observed with a fluorescence microscope Axiovision Z1 by using Apotome system at 40x immersion oil lens (Carl Zeiss, Heidelberg, Germany). Multiple images were taken at different focus distances by using z-stacking (thickness interval: 0.750–1 μm) to localize PKM2 at different focal depths.

Cells were homogenized in RIPA plus buffer [Phosphate Buffered Saline (PBS); NP-40 1%; Na deoxycolate 0.5%; SDS 0.1%; EDTA 1 mM; NaF 50 mM; NaVO₃ 5 mM] containing a cocktail of EDTA-free protease inhibitors (Roche). Protein concentration was determined by the Bradford method by using the BCA Protein Assay Kit (Pierce) and bovine serum albumin as a standard. Twenty micrograms of protein were loaded and subjected to electrophoresis in 10% SDS-PAGE gels (Invitrogen) and transferred onto PVDF membranes (Bio Rad). After 1 h of blocking (LICOR Biosciences) membranes were incubated overnight at 4°C with a rabbit polyclonal anti-PKM2 primary antibody (Cell signaling; 1:1000) or with a mouse monoclonal anti-p53 (Abcam; 1:500). Rabbit monoclonal anti-Actin (1:2000) and mouse monoclonal anti-α-Tubulin antibodies (1:15000) (both from Sigma Aldrich) were used as internal controls. Membranes were incubated with IRDye rabbit and mouse secondary antibodies (1:15000) (LICOR Biosciences) for 45 minutes protected from light. Membranes were scanned by using Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences).