Anti phospho gsk 3α β y279 y216

Anti-phospho-GSK-3α/β (Ser21/9) (#9331), anti-GAPDH (#2118), and β-tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA). Both antibodies were used in a 1:1,000 dilution in TBS containing 3 or 5% BSA (PAA Laboratories, Pasching, Austria). Anti-phospho-GSK-3α/β (Y279/Y216) (#ab68476) and anti-WNT11 (#ab96730) were obtained from Abcam (Cambridge, UK) and used in a 1:1,000 and 1:3,000 dilution in TBS containing 3 or 5% BSA, too. Anti-active-β-catenin (#05-665) was purchased from Merck Millipore (Billerica, MA, USA) and used in a 1:2,000 dilution in TBS and 3 or 5% BSA (Carl Roth, Karlsruhe, Germany).
Anti-Akt (#9272), Anti-Akt (Ser473) (#9271), and Anti-Akt (Thr308) (#9275) were purchased from Cell Signaling (Billerica, MA, USA) and used in a 1:1,000 dilution in TBS and 3% BSA (Carl Roth, Karlsruhe, Germany). The species specific secondary antibodies anti-mouse IgG (whole molecule)-peroxidase (#A9044) and anti-rabbit IgG (whole molecule)-peroxidase (#A0545) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used in a 1:50,000 dilution in TBS containing either 5% BSA or 5% dry milk. Dead and apoptotic cells in response to 1,8-cineol were analyzed by annexin V and propidium iodide staining (Apoptosis Kit II; BD, Heidelberg, Germany).

Anti-phospho-GSK3α/β (Ser21/9) (#9331) and anti-GAPDH (#2118) (Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-GSK3α/β (Y279/Y216) (#ab68476, Abcam, Cambridge, UK) were used in a 1:1000 dilution in TBS containing 3% BSA (PAA Laboratories, Pasching, Austria). Anti-active-β-Catenin (#05-665) (Merck Millipore, Billerica, MA, USA) was used in a 1:2000 dilution in TBS and 3% skim milk (Carl Roth, Karlsruhe, Germany). The species specific secondary antibodies anti-mouse IgG (whole molecule)–peroxidase (# A9044) and anti-rabbit IgG (whole molecule)–peroxidase (#A0545) (Sigma-Aldrich, St. Louis, MO, USA) were used in a 1:50.000 dilution in TBS containing either 3% BSA or 3% skim milk.

Tissue slides were deparaffinized with xylene twice for 10 min each, and rehydrated with alcohol in four baths of decreasing concentrations (100%, 96%, 70% and 50%) for 3 min. each, followed by washing with distilled water and rinsed in TBS three times for 3 min. Antigen retrieval was done using heat induced epitopes retrieval (HIER) microwave oven method. Therefore the slides were cooked in 10 × concentrated citrate buffer (pH 6.0) (DCS Innovative Diagnostik-Systeme, Hamburg, Germany) for 30 min. After cooling down, the slides were covered with 3% hydrogen peroxide for 10 min. to block endogenous peroxidases activity followed by incubation with the specific primary antibodies anti-phospho-GSK3α/β (Y279/Y216) (#ab68476) (Abcam, Cambridge, UK) (1:100) and anti-phospho-GSK3α/β (Ser21/9) (#9331) (Cell Signaling, Danvers, MA, USA) (1:50) over night at 4° C. For negative control, antibody dilution buffer (DCS Innovative Diagnostik-Systeme) was used instead of the primary antibody. The conventional labeled-streptavidin-biotin method with horseradish-peroxidase and 3-amino ethylcarbazole as chromogen was used for detection according to the manufacturer’s instructions. Afterwards the slides were counterstained with Mayer’s Hematoxylin (Carl Roth, Karlsruhe, Germany) and mounted in Faramount Aquaeous Mounting Medium (Dako, Hamburg, Germany).