P gsk3β ser9

Upon biopsy, testis tissues were fixed in Bouin’s solution (Sigma-Aldrich Korea) for 16 hours. After dehydration and clearing, the testis tissues were embedded in Paraplast (Sigma-Aldrich Korea), and 5-µm-thick sections were mounted on poly-L-lysine-coated slides and subjected hematoxylin and eosin (H&E) staining and immunohistochemistry. After deparaffination and rehydration, the slides were blocked in 5% goat serum in PBS. Subsequently, the slides were incubated in a humidified chamber overnight at 4℃ with antibodies specific for GSK3α (#4337; Cell Signaling), GSK3β (#12456; Cell Signaling), p-GSK3α(Ser21) (sc-101690; Santa Cruz), and p-GSK3β(Ser9) (sc-11757-R; Santa Cruz) diluted 1:1,000 in 1.5% goat serum in PBS. After three times washes in PBS, the slides were incubated for 1 hour in goat anti-rabbit IgG H&L (ab6721; Abcam) diluted 1:200 in 1.5% goat serum. After washed three times in PBS, a coloring reaction was performed with ImmPACT DAB substrate (SK-4105; Vector Labs, Burlingame, CA, USA). The nuclei were stained with Harris hematoxylin and permanently mounted with Canada balsam.

All culture media and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-cytokeratin7, -ERα, -PR, -PCNA, -pGsk3β (ser9), -Gsk3β, -c-myc, -β-catenin, -CyclinD1, -Wnt7a, -FZD6, -Dkk-1, -pPI3K(tyr 485), -PI3K, -Akt, -β-actin antibodies, peroxidase- and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were procured from Santa Cruz (Dallas, TX, USA). Antibodies for axin2, pAkt(ser473), cleaved caspase-3 and 9, cleaved PARP, Bax and Bcl-2 were purchased from Cell Signalling Technology, Life Sciences (Boston, MA, USA).
TUNEL detection kit was obtained from Roche (Basel, Switzerland). Immuno-Blot PVDF membrane was purchased from Millipore (Billerica, MA, USA). ECL reagent and ECL Hyperfilm were purchased from GE Healthcare (Little Chalfont, UK).

Western blots were performed as described previously (Venna et al. 2012b (link); White et al. 2012 (link)). Six hours after the onset of cerebral ischemia or a sham surgery with vehicle and GSK-3β inhibitor treatment, the mice were euthanized; brains were rapidly collected and flash frozen. The brain was homogenized in lysis buffer, a BCA assay was performed to determine protein concentrations and an equal amount of protein was loaded on a 4%–15% gradient sodium dodecyl sulfate–polyacrylamide gel and subsequently transferred to a polyvinylidene difluoride membrane. p-GSK-3β (Ser-9) (1:2500;Santa Cruz Biotechnology), TAK1 (1:500; Cell Signaling Technologies), β-Catenin (1:1000; Cell Signaling Technologies); β-actin (1:5000; Sigma) was used as the loading control. Blots were incubated overnight in primary antibody at 4°C in Tris-buffered saline containing 4% bovine serum albumin in 0.1% Tween20. Secondary antibodies (goat anti-rabbit IgG 1:10,000 and goat antimouse IgG; Chemicon) were diluted and incubated for 45 min at room temperature, ECL (Pico) detection kit (ThermoScientific) was used for signal detection. Densitometry was performed using Adobe Photoshop software.