Gli1 sc 20687

HPAC cells (1 × 10⁴ cells/well) were seeded in 8-well chamber slides and treated after 24h with 25μM gedunin, 1μg/ml rhShh, 5μM GANT-61, 1μg/ml rhShh and 25μM gedunin, 5μM GANT-61 and 1μg/ml rhShh. Then 24h after incubation with the respective treatments the cells were fixed with 100% methanol and 100% acetone (ratio 1:1). Membrane permeabilization was done using 0.2% Triton X in PBS for 20 min and blocked with 5% BSA. Slides were then incubated with Shh (sc-9024) and Gli1 (sc-20687) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24h followed by Alexa fluor 488-conjugated anti–rabbit secondary antibody (Life Technologies, Grand Island, NY, USA). Images were captured by Nikon laser scanning confocal microscope.

The skin, corneal epithelium, lung tissue or cultured cells were extracted with cold lysis buffer comprising 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P‐40, 0.5% sodium deoxycholate, 0.1% SDS, and protease and phosphatase inhibitor cocktails. Equal amounts of protein extracts were subjected to electrophoresis on 8% or 10% SDS‐PAGE and then electrophoretically transferred to PVDF membrane. After blocking in 1% BSA for 1 hr, the membranes were incubated with primary antibodies ZO‐1 (61‐7300, 1:1000, Invitrogen), claudin‐1 (519,000, 1:1000, Invitrogen), Gli‐1 (sc‐20687, 1:500,Santa Cruz) and β‐actin (A3854, 1:10,000, Sigma‐Aldrich) overnight at 4°C. After three washes with Tris‐buffered saline with 0.05% Tween‐20 for 10 min. each, the membranes were incubated with HRP‐conjugated goat or rabbit anti‐mouse or rabbit IgG (1:10,000) for 1 hr. The results were visualized by enhanced chemiluminescence reagents and recorded with an imaging system (ChemiDoc XRS, Bio‐Rad, Hercules, CA, USA).