Ab14234

HeLa cells were reseeded 24 h post-transfection on 12 mm glass coverslips (Fisher Scientific). The next day, cells are fixed for 20 min with 4% EM-grade paraformaldehyde (PFA; Electron Microscopy Sciences 15710) and 4% sucrose in PBS at room temperature and washed three times in PBS.
To visualize the plasma membrane, cells were incubated with wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (1:200, Invitrogen w11262) for 10 min at room temperature and washed twice with PBS. Afterwards, the nuclei were stained with Hoechst 33342 (1:2,000; Invitrogen H3570) for 10 min at room temperature and washed twice with PBS. Coverslips were mounted using fluorescent mounting medium (Dako S302380) and stored at 4 °C before being subjected to fluorescence microscopy.
To visualize the ER, fixed cells were permeabilized with 0.5% Triton-X in PBS for 5 min. After washing with PBS, coverslips were blocked with 3% BSA in PBS for 1 h at room temperature. Next, cells were incubated for 2 h with a primary chicken anti-calreticulin antibody as a marker for the ER (1:500, Ab14234, Abcam, USA), followed by three washes with PBS and staining with an Alexa Fluor 594® conjugated goat anti-chicken IgY secondary antibody (1:500, A-11042, Thermo Fisher Scientific, USA) for 1 h. Nuclear staining and mounting were performed as described above.

Monoclonal anti-Wnt-3a and anti-β-catenin were described previously41 (link). Rabbit polyclonal antibody against Wnt11 (ab31962) and chicken polyclonal antibody against Calreticulin (ab14234) were purchased from Abcam. Mouse anti-Tsg101 (612697), anti-CD81 (555675), and anti-Flotillin-2 (610383) antibodies were purchased from BD Transduction Laboratories. Rabbit polyclonal anti-CD63 antibody (orb11597) was obtained from Biorbyt. Western blotting was performed according to a standard protocol. Mouse anti-Hsp70 (H5147) antibody was purchased from Sigma.