Anti hemagglutinin ha

Anti-phospho-Akt, anti-Akt, anti-phospho-p70S6K and anti-hemagglutinin (HA) antibodies were purchased from Cell Signaling Technology, Inc., and used at a dilution of 1:1,000, according to the manufacturer’s instructions. The JC12 anti-FOXP1 antibody was provided by Professor Alison H. Banham and used at a dilution of 1:30. β-actin mouse monoclonal antibody (dilution, 1:5,000) and horseradish peroxidase-conjugated anti-mouse antibody (dilution, 1:2,500) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Agilent Technologies, Inc. (Santa Clara, CA, USA), respectively.

Tissue samples were dissected in PBS, and then fixed in 4% formaldehyde in PBS for 20 min. After washing with PBS with 0.2% Triton X-100 (PBST), the samples were incubated in PBT with primary antibodies at 4°C overnight with shaking, and then washed in PBT three times for 15 min each. The following antibodies were used in immunostaining: anti-GFP (1:200; Cell Signaling Technology, #2956S), anti-hemagglutinin (HA) (1:500; Cell Signaling Technology, #3724S), anti-HNF4 guinea pig polyclonal antibody (1:100; a gift from G. Storelli) (65 (link)), anti-Fru^COM rabbit polyclonal antibody (1:500), and rabbit anti-Fru^M polyclonal antibody (1:250) (59 (link)). The secondary antibodies conjugated with Alexa Fluor 546 or 633 (Invitrogen) were diluted 1:200 and incubated at room temperature for 2 hours. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (1:1000; Invitrogen, #D1306). After washing, samples were mounted and imaged with Zeiss LSM 800 or Zeiss LSM 980 Confocal Microscopes. Image analysis was performed in ImageJ.