Anti gli1 antibody

Fresh mesothelioma tissues were obtained from patients who were undergoing surgical resection of the primary tumor. All human tissue samples were obtained and analyzed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714-22 942–01). The tissue microarray sections were immunostained as previously described [33 (link)]. Anti-CK2α antibody was from Millipore (Billerica, MA). Anti-Gli1 antibody was from Cell Signaling (Beverly, MA). The following scoring system was used: −, no stain; +, weak staining (10% or above stained cellularity considered as positive); ++, moderate staining (30% or above stained cellularity considered as positive); +++, strong staining (50% or above stained cellularity considered as positive). All scoring was done under low power objective lens (10×) with a Zeiss Axioscop 2 microscope (Carl Zeiss Inc, Germany). Images were taken under 10x or 40x objective lens.

After treated and untreated U87 and T98G cells lysate were harvested, equivalent total protein was separated in 7.5% (10%, 12.5%) sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membrane (Millipore, USA). The membrane was blocked in 5% milk (5% BSA)–TBST solution at room temperature, and incubated separately with anti-SHH antibody, anti-SMO antibody, anti-GLI1 antibody, anti-Bcl-2 antibody, anti-Foxm1 antibody, anti-Bax antibody, anti-Caspase-3 antibody, anti-PARP antibody, anti-ATM antibody, anti-p-ATM antibody, anti-γH2AX antibody, anti-N-cadherin antibody, anti-β-catenin antibody, anti-Vimentin antibody, anti-E-cadherin antibody, anti-Histone H3 (D1H2) antibody (all from Cell Signaling Technology, USA), anti-NF-κB (p65) antibody, anti-p-NF-κB (p-p65) antibody (Wanleibio, China), anti-β-actin antibody, anti-MGMT antibody (Santa Cruz, USA) and anti-GAPDH antibody (Sigma, USA). Following incubation in HRP labeled secondary antibody (Introvigen), protein bands were scanned with the ECL system and detected by Gel Doc 2000 (Bio-Rad).