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2 protocols using auy922

1

Antibody Characterization for Protein Analysis

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Anti-human TRPM1 (F-3, western blotting 1: 250; immunoprecipitation 1:50; immunohistochemistry 1:100), anti-HSP70 (W27, for western blotting, 1:1000), anti-HSP90 α/β (F-8, for western blotting, 1:1000), anti-CDC37 (C-11, for western blotting, 1:1000) antibodies, and anti-HSP90 α/β conjugated agarose were from Santa Cruz Biotechnology. Anti-mouse Trpm1 (western blotting 1:200; immunohistochemistry 1:100; immunofluorescence 1:100) was from Novus Biologicals. Anti-cleaved caspase 3 (Asp175) (western blotting 1:1000; immunohistochemistry 1:100), anti-AKT (western blotting 1:1000; immunohistochemistry 1:100), anti-phospho-AKT(Ser473) (D9E, immunohistochemistry 1:100), anti-β-Actin (13E5, western blotting 1:2000; immunofluorescence 1:500), anti-PARP1 (# 9542, western blotting 1:1000) and anti-Ki67 (D2H10, immunohistochemistry 1:100) antibodies were purchased from Cell Signaling Technology. Anti-Rhodopsin (Rho 1D4, immunohistochemistry 1:100), anti-RPE65 (immunofluorescence 1:250), anti-FLAG M2 (western blotting 1:1000) antibodies and anti-FLAG M2 affinity agarose gel were from Sigma-Aldrich. Anti-GFAP antibody (immunohistochemistry 1:100) was from Dako. Biotinylated Peanut Agglutinin ((immunohistochemistry 1:500) was obtained from Vector Laboratories. AUY922 and MG132 were from MedChem Express.
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2

Cellular Proliferation Assay with Inhibitors

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Primary cell line CF-00034-5 was prepared in a 384-well plate for cellular proliferation assay in an identical manner as above. After overnight incubation, the cultures were treated with inhibitors BKM120 (HY-70063, MedChemExpress LLC), BYL719 (S2814, Selleck Chemical), and AUY922 (HY-10215, MedChemExpress). All of the pharmaceuticals were administered at final concentrations of 0.3, 0.949, 3, 9.49, and 30 µM. Cellular proliferation was measured by Cell Titer Glo as described above. IC50 values were determined using GraphPad software and combination indexes were calculated using CalcuSyn software (Biosoft).
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