Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts
Figure 2
Construction and transduction of of pTAT-mcMyc recombinant protein to MEF in vitro. (a) Schematic representation of the pTAT-mcMyc construct. (b) Reduced and denatured pTAT-mcMyc recombinant protein was resolved on 12–15% SDS-polyacrylamide gel and transferred to PVDF membrane by electrophoresis (90 V, 2 hours, 4°C). Wet membrane was blocked for 1 hour in Odyssey blocking buffer (1 : 1 Tris-HCl buffer) and probed with either mouse anti-6xHistidine or mouse anti-mouse cMyc primary antibody before detection with goat anti-mouse Alexa Fluor-680 secondary antibody. Recombinant pTAT-mcMyc was detected at predicted molecular weight (57–60 kDa; arrow). (c) MEFs were plated to coverslips before 100 nM pTAT-mcMyc recombinant protein was applied to equilibrated culture media. Following a 1-hour incubation, MEFs were washed, fixed, blocked, and probed with antibody recognizing 6xHistidine (bottom panel). Cell nuclei were detected with Hoechst dye (middle panel). Scale bar: 200 μM. Red arrows shown detection of pTAT-mcMyc protein outside of detectable nuclei. (d) MEFs were infected with retrovirus harboring OSK or left uninfected. OSK-expressing MEFs were treated with 100 nM pTAT-mcMyc protein (red square) or pTAT-control protein (green triangle) daily for 12 days. Uninfected MEFs were left as a control, or treated with 100 nM pTAT-mcMyc protein (brown diamond) daily for 12 days. On days 5, 7, 9, and 12 after infection, Oct4-GFP+ colonies were counted in duplicate wells. Mean ± SEM of independent experiments shown; .
