C myc antibody 9e10

pCDNA3 plasmid harbouring Myc-AMPKα1(WT), Myc-AMPKα1(S173C) or Myc-AMPKα1(S173D) [39 (link), 40 (link)], kindly given by Dr. Dietbert Neumann (Maastricht University, The Netherlands), were used to generate populations of C3A cells which stably express mutated forms of the AMPKα1(S173) residue. C3A cells were transfected by electroporation, as previously described [37 (link)]. After 24 h, the antibiotic Geneticin (500 μg/ml) (Invitrogen, Thermo Fisher Scientific) was added to the media in order to select the transfected cells. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

Cultured cells were grown on sterile glass cover slips and fixed with 3.7% paraformaldehyde at room temperature for 20 minutes. Cells were permeabilized by 0.1% saponin for 10 min and blocked with 10% donkey serum in PBS (Invitrogen, UK) for 20 minutes at room temperature. The cells were stained with the following primary antibodies: c-Myc Antibody (9E10) (Santa Cruz; 1:200), p53 (B-P3, Santa Cruz, 1:200), Caspase-3 Antibody (Cell Signaling; 1:400) or AlexaFluor 488 phalloidin (Life Technologies, 1:25). The corresponding secondary antibodies, AlexaFluor 488- and 555-conjugated donkey anti-mouse and anti-rabbit IgG (Invitrogen) were used at a concentration of 5 μg/mL, at room temperature for 1 hour. The cells were washed with PBS. Nucleic acid was stained with 0.5 μg/mL Hoechst stain (# 33342). The cover slips with cells were dropped onto a glass slide with mounting medium (Sigma-Aldrich, St. Louis, MO) and visualized with Zeiss Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY). An ORCA-ER Digital Camera (Hamamatsu) was used for visualization and microphotography. The caspase-3 activity in detached cells was measured using using Image-iT Live green caspase detection kit (Life Technologies), as recommended by the manufacturer.