Anti ptch

The proteins were isolated from the ischemic cortex tissue using lysis buffer (Beyotime, China), and protein concentrations were examined using a BCA protein assay kit (Beyotime, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies: anti-Shh, anti-Ptch, anti-Smo, anti-Gli1, anti-CD34 (1:1000, Santa Cruz Biotechnology, USA), and anti-VEGF (1:1000, Abcam, UK) overnight at 4 °C. After rinsing four times in TBST buffer for 5 min, the membranes were incubated with secondary antibodies (1:20000, ZSGB-BIO, China) at room temperature for 2 h and then treated with ECL reagent (ThermoFisher, USA) to detect protein expression levels. The protein bands were quantitatively analyzed using the Image J software (Rawak Software Inc., Germany).

Genipin was purchased from Calbiochem (San Diego, CA, USA). GANT61 was purchased from Selleckchem (Houston, TX, USA). Protein G PLUS-Agarose and anti-BAX, anti-BCL2, anti-MCL-1, anti-UB, anti-BCL-Xl, anti-GLI3, anti-PCAF, anti-SHH, anti-PTCH, and anti-p53 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-PUMA, anti-BIM, anti-SURVIVIN, anti-BID, anti-cleaved PARP, anti-CASP3, anti-CASP9, anti-β-TrCP, and anti-GLI1 antibodies were purchased from Cell Signaling (Beverly, MA, USA). The anti-actin antibody was obtained from Sigma (St. Louis, MO, USA), the anti-SMO antibody was obtained from Abcam (Cambridge, UK), and the anti-ITCH antibody was purchased from BD science (San Jose, CA, USA). The secondary antibodies, anti-mouse IgG HRP and anti-rabbit IgG HRP, were obtained from Cell Signaling.