Anti hsp 90α β f 8

Preparation of whole cell lysates and western blot analysis were performed as previously described [51 (link)]. The following primary antibodies were used at 1:1000 dilution: anti-E-cadherin (32A8; Cell Signaling, Frankfurt, Germany), anti-HSP 90α/β (F-8; Santa Cruz, Heidelberg, Germany), anti-L1CAM (9.3; kindly provided by Prof. G. Moldenhauer, German Cancer Research Center, Heidelberg, Germany), anti-Zeb1 (Novus Biologicals, Wiesbaden-Nordenstadt, Germany). Anti-Vimentin (V9; Santa Cruz) was applied 1:200 and anti-phospho-p65 (12H11; Merck Millipore) 1:500. HSP90 was used as loading control. Secondary antibodies anti-mouse IgG (HRP-linked) and anti-rabbit IgG (HRP-linked) (both Cell Signaling) were used at 1:2000 dilution. Primary antibodies were incubated at 4°C overnight, while secondary antibody incubation was performed for 1 h at room temperature (RT). Blots were incubated in Clarity Western ECL Substrate (Bio-Rad Laboratories, München, Germany) and visualization of proteins was performed by using the Fusion SL detection system (Vilber Lourmat, Eberhardzell, Germany).

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.