Odyssey membrane scanning system

Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The protein containing PVDF membranes were probed with different primary antibodies: anti-PAK2 (2615, Cell Signaling Technology, Beverly, MA, USA), anti-c-Myc (9402, Cell Signaling Technology, Beverly, MA, USA), anti-CCND1 (ab134175, Abcam, Melbourne Australia), anti-PKM2 (4053, Cell Signaling Technology, Beverly, MA, USA), anti-Active β-Catenin (05665, Millipore, Burlington, USA), anti-GAPDH (5174, Cell Signaling Technology, Beverly, MA, USA), anti-β-Catenin (9562, Cell Signaling Technology, Beverly, MA, USA) and anti-flag (NBP1-06712SS, Novus Biologicals, Littleton, CO, USA) in a 1:1000 dilution. After 2 h of primary antibody incubation at room temperature, membranes were washed with 1× TBST (tris-buffered saline and Tween-20) and incubated with secondary antibodies Alexa-Flour 680 anti-rabbit IgG (A21109, Thermo Fisher Scientific, Waltham, MA, USA) and Alexa-Flour 790 goat anti-mouse IgG (A28182, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. The membrane was washed, and bands were visualized using an Odyssey membrane Scanning system (Li-Cor Biosciences, Bad Homburg, Germany).

Cells were pelleted and lysed with urea lysis buffer (8M Urea: Sigma IU5378, 2M Thiourea: Sigma 11149, 10% CHAPS: Sigma C9426, 10% Dithiothreitol: Sigma D9779) and kept at 4°C for 30 min. After incubation, the lysate was centrifuged at 14,000 rpm for 2 h, and the supernatant was collected in a 1.5 ml microcentrifuge tube and stored at –80°C. The proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The protein-containing PVDF membrane was blocked with 5% non-fat milk in tris buffered saline with 0.05% Tween 20 (TBST) for 30 min. The membrane were then probed with following primary antibodies: anti-SRSF10 (Sigma, HPA053831), anti-pERK (CST, 9101S), anti-ERK (CST, 9102S), anti-EGR1 (CST, 4154S), anti-flag (Novus Biologicals, NBP1-06712SS), anti-PKM2 (CST 4053S), anti-PKM1 (CST 7067S), and anti-GAPDH (CST 5174S). Anti-GAPDH was used as loading controls for protein assays. After 2 h of incubation with primary antibody at RT, membranes were then washed with 1× TBST then again incubated with secondary antibodies for 45 min at RT. The probed PVDF membranes were washed with TBST, and the bands were visualized using an odyssey membrane scanning system (Li-cor Biosciences, Bad Homburg, Germany).

The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The protein-containing PVDF membranes were then probed with following primary antibodies: Anti- PKM1 (Cell Signaling Technology,7067S), Anti-PKM2 (Cell Signaling Technology, 4053S) to identify the level of PKM isoform, Anti- BORIS (Millipore ABE631), AntiDNMT3B (Abcam, ab13604), anti-flag (Novus Biologicals, NBP1-06712SS) and Anti GAPDH (Cell Signaling Technology, 5174S) were used as loading controls for protein assays. After 2 h incubation with primary antibody at room temperature (RT), membranes were washed with 1X tris-buffered saline and Tween-20 (TBST) then again incubated with secondary antibodies for 45 min at RT. The probed PVDF membranes were washed, and the bands were visualized using an Odyssey membrane Scanning system (Li-Cor Biosciences, Bad Homburg, Germany).