Stella 3200

Total protein extracts were prepared by lysing frozen PDX tumor cells in FastPrep lysing matrix tubes (MP Biomedicals, Illkirch, France) with lysis buffer containing cOmplete protease inhibitor cocktail, PhosSTOP phosphatase inhibitor (Roche, Mannheim, Germany). 30 μg protein were separated by SDS-PAGE and transferred onto nitrocellulose membranes. For immunoblotting, membranes were probed with primary antibodies overnight at 4°C, then incubated with anti-rabbit IgG-HRP (Cell Signaling). Signals were detected by enhanced chemiluminescence (SuperSignal WestDuraExtendedDurationSubstrate, Thermo Fisher Scientific) using CCD camera STELLA3200 (Raytest, Straubenhardt, Germany). Antibodies were used at a dilution of 1:1000, except anti-β-actin (loading control; 1:10000).

SDS- polyacrylamide gel electrophoresis (PAGE) was used to separate protein samples according to their electrophoretic mobility [56 (link)]. The separated proteins were stained with Coomassie Brilliant Blue R250 or electro-blotted on a polyvinylidene difluoride membrane (Roth) with a semi-dry blotter (Bio-Rad) [57 (link)]. For Western blot analyses, antibodies directed against the His-tag (monoclonal, horseradish peroxidase (HRP)-conjugated, Novagen, 1:1000), the cyt. b₆ N-terminus (SKVYDWFEER, polyclonal, rabbit, Gramsch Laboratories, Munich, Germany, 1:10000) or the cyt. b₆ C-terminus (EIRKQGISGPL, polyclonal, rabbit, Gramsch Laboratories, Munich, Germany, 1:10000) were used. In case of the latter ones, anti-rabbit antiserum (HRP-conjugated (1:10000), Sigma-Aldrich) was used as a secondary antibody. If HRP-conjugated antibodies were used, membranes were developed using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc.), and chemiluminescence was detected using a Stella 3200 (Raytest).

Modioli including the SGNs were isolated from Ano2 knockout mice and their wildtype littermates (P5). For immunoblots, the tank blotting Mini-Protean Tetra system (Bio-Rad) and transfer buffer with methanol (25 mM Tris, 192 mM glycine) were used. 10 µg of proteins were separated in 5% acrylamide gels. Proteins were blotted on polyvinylidene membranes (Roti-PVDF, Roth). Protein transfer was controlled by staining with Ponceau S (0.2% Ponceau S, 3% acetic acid). Blocking was done in Tris buffered saline (TBS) supplemented with 0.05% Tween-20 and 5% dry milk powder. TMEM16B was detected using the same antibody as described in the immunohistochemistry section (1:1000). It was incubated overnight at 4°C in TBS-T with 1% dry milk powder. Washing was done with TBS/0.05% Tween-20. The secondary antibody (goat anti-guinea pig IgG antibody-HRP, Merck, 1:1000) was incubated at RT for 2 hr in TBS-T with 1% dry milk powder. Signals were visualized by chemiluminescence (Amersham ECL Prime Western Blotting Detection Reagent, GE Healthcare). Documentation was done with the camera system Stella 3200 (Raytest) and the Xstella software.

Snap-frozen cells were homogenized in lysis buffer (50mM TRIS-HCl pH 7.6, 250mM NaCl, 5mM EDTA, 0,1% Triton X-100) containing cOmplete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor (Roche, Mannheim, Germany) by sonication. Immunoblotting was performed as previously described (35 (link)). Antibodies were used at 1:1.000 dilution, except antibodies against CRMP2, phospho-Stathmin (1:500); phospho-AKT, phospho-ERK1/2, phospho-RPS6 (1:2.000); β-Actin (1:10.000). Luminescent signals were detected with the digital gel documentation system Stella3200 (Raytest, Straubenhardt, Germany) and quantified using ImageJ 152-win-java8 software (National Institutes of Health, USA).

For quantitative evaluation of gene expression levels, capillary Northern blot technique was applied according to the protocol for transfer onto nylon membranes at neutral pH, published in Sambrook and Russell (2001) . Subsequent labeling of specific RNA molecules was performed with digoxigenin-11-UTP (DIG-dUTP, Roche Diagnostics Deutschland GmbH, Mannheim, Germany)-labeled probes, generated by PCR (Supplementary Table S3). Labeled RNA was detected using anti-DIG antibodies and the substrate CDP-Star (both obtained from Roche Diagnostics Deutschland GmbH, Mannheim, Germany). Visualization and quantification was achieved by chemiluminescence using the imaging system Stella 3200 and AIDA imaging software (raytest, Straubenhardt, Germany).