Anti e cadherin 32a8

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).

Cells grown confluent on cover slips were fixed in 4.5% paraformaldehyde for 10 min and permeabilized with methanol for 10 min at −20°C. Blocking was performed using 4% Bovine Serum Albumin (BSA, Serva Electrophoresis, Heidelberg, Germany) in PBS supplemented with 0.3% Triton X-100 (Sigma-Aldrich) for 1 h at RT, before primary antibody incubation was performed at 4°C in a humidified chamber overnight. Antibodies were diluted in 1% BSA/PBS/0.3% Triton X-100 as follows: anti-Nanog (D73G4; Cell Signaling) 1:100, anti-Nestin (10C2; Thermo Fisher Scientific) 1:200 and anti-E-cadherin (32A8; Cell Signaling) 1:50. After washing, incubation with fluorochrome-labeled secondary antibodies (AlexaFluor 488 anti-mouse; AlexaFluor 546 anti-rabbit (both Life Technologies, Carlsbad, USA), diluted 1:500 in 1% BSA/PBS/0.3% Triton) and 2 μg/ml Hoechst 33258 (Sigma-Aldrich) for nuclear staining was performed for 1 hour at RT before samples were mounted in FluorSave Reagent (Merck Millipore) mounting medium. Control staining was performed in parallel with isotype control antibodies (Isotype control mouse IgG1 (MAB002; R&D Systems, Minneapolis, USA); Isotype control rabbit IgG (SP137; abcam, UK)) and showed no or only weak staining.