Af647 ctxb

Cells were fixed with 4% PFA for 20 min at room temperature, or with methanol for 10 min at −20 °C, then cyto-centrifuged onto coverslips. Fixed cells were permeabilized and blocked for 30 min in PBS supplemented with 0.1% saponin and 3% bovine serum albumin, and then incubated with a primary and a secondary antibody for 1 h each. To stain with anti-Akt(pSer473) (Cell Signaling Technology; 193H12), 10% skimmed milk was used for blocking. After washing with PBS, cells were mounted with Fluomount (DiagnosticBioSystems). For staining endocytic compartments, cells were incubated for 1 h with 5 μg ml⁻¹ AF647-CTXB or 1 mg ml⁻¹ AF647-dextran (Molecular Probes). Confocal images were obtained by a Fluoview FV10i laser scanning microscope with an x60 1.20 N.A. water-immersion objective (Olympus). Composite figures were prepared with Photoshop elements 10 and Illustrator CS6 software (Adobe). Pearson’s correlation coefficients (Pearson’s R) were calculated with NIH ImageJ Version 1.48v software.

Fluorescent-tagged cholera toxin B subunit (AF647-CTXB), bovine serum albumin (AF647-BSA) and transferrin (AF633-Tfn) were obtained from Molecular Probes (Life Technologies, Grand Island, NY, USA). HepG2 cells were incubated with a mixture of AF488-RNP (1 pmol/ml) and AF647-BSA or AF633-Tfn (10 µg/ml) for 1 h at 4°C. Alternatively, cells were incubated with AF488-RNP for 45 min at 4°C before addition of CTXB and continuation for another 15 min. Cells were then transferred to 37°C for the indicated time before observation.