Anti oct4 h 134

Cells were lysed in RIPA buffer (0.1% SDS, 50mM Tris-HCl pH8.0, 150mM NaCl, 0.5mM EDTA, and 1% NP-40) supplemented with a cocktail of protease inhibitors (Sigma). Cellular extracts were quantified for protein using BCA assay kit (Thermo Scientific Pierce, Rockford, IL), subjected to SDS-PAGE and electrotransferred to polyvinylidine difluoride membrane. For immunoblotting assays, anti-Nox4 [20 (link)], anti-Oct4 (H-134; Santa Cruz Biotechnology), anti-Sox2 (Millipore), anti-Klf4 (Santa Cruz Biotechnology), anti-c-Myc (Cell Signaling Technology, Danvers, MA), anti-β-Actin (Santa Cruz Biotechnology), anti-p-AKT (Cell Signaling Technology), anti-AKT (Cell Signaling Technology), anti-p-Erk-1,2 (Cell Signaling Technology), anti-Erk-1,2 (Cell Signaling Technology) and anti-α-tubulin (Sigma) antibodies were used. A horseradish peroxidase-conjugated secondary antibody (Invitrogen) was applied prior to visualization by chemiluminescence (Amersham Imager 680; GE Healthcare Life Sciences, Chicago, USA).

Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, and permeabilized in 0.5% Triton X-100 for 10 min. After blocking with PBS containing 1% bovine serum albumin for 1 h at room temperature, the cells were incubated with the following primary antibodies: anti-βIII-tubulin (Millipore), anti-smooth muscle actin (SMA; Dako, Glostrup, Denmark), anti-α-fetoprotein (Dako), anti-Oct4 (H-134; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox2 (Millipore), anti-SSEA1 (Santa Cruz Biotechnology) antibodies overnight at 4℃. The secondary antibodies, Alexa Fluor 594-conjugated goat anti-mouse IgG (Life Technologies, Grand Island, NY), Alexa Fluor 594-conjugated goat anti-rabbit IgG (Life Technologies), Alexa Fluor 488-conjugated goat anti-mouse IgG (Life Technologies) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies) were applied for 1 h at room temperature in the dark. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and examined with LSM 880 with Airyscan (Carl Zeiss, Jena, Germany).