Pkm2 antibody

Cell lines plated on glass bottom dishes (35 mm; MatTek Corp.) and dispersed islets plated on glass bottom plates (48 well; MatTek Corp.) were cultured on laminin and/or transduced with viral constructs for 24 h. Once fixed with 100% ice-cold methanol (-20 °C; 15 min), samples were blocked for 1 h in 5% goat serum/PBS at RT. Samples were consequently incubated for 24 h (4 °C) with FGFR5 antibody (Invitrogen; #PA5-21,516) diluted 1:1000 in 1.5% goat serum/PBS, or PKM2 antibody (Cell Signalling Technology; #3198) diluted at 1:500 in 1.5% goat serum/PBS. The antibodies were subsequently detected using anti-rabbit Alexa Fluor 560 diluted at 1:1000 in block solution (5% goat serum/PBS; RT for 45 min). Nuclei were counterstained for 3 min at RT with DAPI (5 µg/ml). All samples were imaged using the 63 × /1.4–NA oil-immersion objective lens of the ASI wide field microscope.

After treatment at the indicated time, cells were harvested, and the proteins from cells were separated by SDS–PAGE in 12% (w/v) polyacrylamide gels and transferred to PVDF membranes. The membranes were then incubated with the appropriate primary antibody overnight at 4 °C. After subsequently rinsing in PBS, membranes were incubated with secondary antibody conjugated to horseradish peroxidase, the bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, San Jose, CA, USA), and the blots were exposed to film (Thermo Fisher Scientific, San Jose, CA, USA) [28 (link)]. Flag antibody (Sigma, St. Louis, MO, USA), HA antibody (Abcam, Cambridge, MA, USA), PKM2 antibody (Cell Signaling Technology, Danvers, MA, USA), HNF4α antibody (Santa Cruz, CA, USA) were used for protein assay.

The RPMI-1640 medium, DMEM-F12 medium and heat-inactivated fetal bovine serum (FBS) were from GIBCO (Gaithersburg, MD, USA). The RNAiso Plus was purchased from Takara (Shiga, Japan). TransScript First-Strand cDNA Synthesis SuperMix and TransStart Top Green qPCR SuperMix were purchased from TransGen (Beijing, China). PKM2 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies for p53, Drp1 and ETC complexes were obtained from Proteintech (Chicago, USA). Antibody against GAPDH was from Bioworld Technology (Minneapolis, MN). Actinomycin D (RNA synthesis inhibitor), Cycloheximide (CHX, protein synthesis inhibitor) and MG-132 (the proteasome inhibitor) were obtained from Sigma (St. Louis, USA).

For immunofluorescence staining, cells cultured on glass-bottom culture dishes were washed twice with PBS, and then fixed with 4% paraformaldehyde for 1 h. After fixation, cells were permeabilized for 10 min in 0.5% Triton X-100 (Cat# ST1723, Beyotime) in PBS, and then blocked in blocking buffer (Cat# P0260, Beyotime) for 1 h at room temperature. PKM2 antibody (Cat#4053, Cell Signaling) diluted in primary antibody dilution buffer (Cat#P0262, Beyotime) was used to incubate the cells overnight at 4 °C. Primary antibodies used in this assay are the same with antibodies used in western blotting. Subsequently, cells were washed 3 times with PBS, and then incubated with a secondary antibody Alexa Fluor 488 Polyclonal Antibody (Cat#A-11094, Invitrogen) for 1 h at room temperature. DAPI (Cat# C1005, Beyotime) was used to counterstain the nuclei after cells were washed 3 times with PBS. Fluorescent images were acquired using the Echo Revolve Hybrid optical microscope.
To determine the viability of cultured cells, crystal violet staining was performed as previously described [17 (link)]; cells cultured on 6-well plate were fixed as described above, washed twice with PBS, and then stained with crystal violet staining solution (Cat# C0121, Beyotime) for 10 min; and, after washing 3 times with PBS, bright-field images were acquired using the Echo Revolve Hybrid optical microscope.