Anti smurf2

Cells at 60% confluence were plated onto sterilized glass coverslips. The slides were washed in phosphate-buffered saline (PBS) and fixed for 20 min in ice-cold acetone/methanol (1:1) on ice. The slides were then blocked with 3% BSA in PBS for 1 hour at 37°C, followed by incubation with anti-Smurf2 (25511, Santa cruz Biotechnology), anti-Ki67 (H300, Santa cruz Biotechnology), FITC-conjugated anti-rabbit, and PE-conjugated anti-mouse (2012, Santa cruz Biotechnology) antibodies. Cells were also stained with Hoechst dye (Hoechst 33342, Invitrogen) to reveal nuclei. The images were taken at × 60 magnification using a confocal microscope (FV1000 Olympus).

HPMCs were grown on cover-slips, washed twice with PBS, fixed in 4% paraformaldehyde for 20 min and permeabilized using 0.1% Triton X-100. Anti-ZO-1 (1:100 dilution, Santa Cruz), anti-Vimentin (1:100, Santa Cruz), anti-pAkt (1:80, Cell Signaling) and anti-Smurf2 (1:80, Santa Cruz) antibodies were diluted in a blocking buffer and incubated with cells overnight at 4°C. The cover-slips containing the cells were washed with PBS 3 times, followed by incubation with FITC-labeled secondary antibodies (Santa Cruz) for 2 hrs at 22°C. The cells were then examined using a fluorescence microscope.

Western blot analyses were performed as described previously [7 (link)]. Briefly, samples (20 μg protein) were subjected to SDS–PAGE. The proteins were transferred onto nitrocellulose membranes, which were probed with the specific antibodies: anti-ZO-1, anti-vimentin, anti-Smad7 and anti-Smurf2 (Santa Cruz, 1:1000), anti-pan-Akt (phospho T308) antibody (Abcam) also known as anti-pAkt, anti-total Akt, (Cell Signaling, 1:1000) and anti-FN (BD Biosciences, 1:1000). A peroxidase-conjugated goat anti mouse IgG (1: 10 000) was used as a secondary antibody. For detection of other proteins or β-actin, the membranes were treated with Stripping buffer and then re-probed with another antibody or β-actin antibody, the latter served as an internal control.