Plan apo 63 1

For p50, p150glued and pAKT immunofluorescence, cells were fixed in 3% paraformaldehyde followed by permeabilization with 0.2% Triton‐X‐100 for 10 min. Nuclei were visualized using Hoechst dye (33258; Sigma‐Aldrich). Coverslips were mounted on glass slides using ProLong Gold Antifade (Invitrogen). Cells were visualized with an Axiovert 200 inverted microscope (Carl Zeiss, Inc.) using Plan‐Neo 100×/1.30 or Plan‐Apo 63×/1.40 oil‐immersion objectives (Immersol 518F; Carl Zeiss, Inc.). In some cases, optical sections were deconvolved using axiovision combined iterative algorithm to obtain confocal images.

Cells were plated onto 12-mm glass coverslips in 24-well plates before drug treatment or transfection. For Figures 1, 2, and 3, D and E, and Supplemental Figures S1 and S2, cells were cultured for 12 h in medium without FBS or ITS in order to obtain maximum sensitivity to insulin (starved). Then, 1% ITS (Cellgro) or 3 µM CT99021 (Cayman Chemicals) was added for the indicated times. For Figures 3, A–C, and 7, A–C, after starvation, cells were exposed to 3 µm CT or 10 μM rosiglitazone (Biomol) for 12 h in full medium (containing both ITS and FBS). Cells were fixed and then processed for IF. For DIC, APC, or Ndel1 antibodies, cells were fixed in100% ice-cold methanol for 2 min; for tubulin and AKT antibodies cells, were fixed in warm 4% paraformaldehyde for 20 min followed by permeabilization with 0.2% Triton X-100 for 10 min. Nuclei were visualized using Hoechst dye (33258; Sigma-Aldrich). Coverslips were mounted onto glass slides using ProLong Gold Antifade (Invitrogen). Cells were visualized with an Axiovert 200 inverted microscope (Carl Zeiss) using Plan-Neo 100×/1.30 or Plan-Apo 63×/1.40 oil-immersion objectives (Immersol 518F; Carl Zeiss) or a Plan-Neofluor 20× dry objective.