Phospho erbb2

Protein extraction and Western blot (WB) were performed according to classical procedures. To detect OCAM in the medium, 10 ml of neurosphere-conditioned supernatant was syringe-filtered (10 μm), precipitated with 4 vol. of cold acetone (-20°C) and resuspended in 200 μl Laemmli loading buffer. Antibody references and dilutions are: rabbit OCAM ([13 (link)]; 1:500), goat OCAM (R&D, #AF778, 1:2000), ErbB2 (Cell Signaling, #2165, 1:1000), phospho-ErbB2 (Cell Signaling, #2241, 1:1000). To detect phosphorylated receptors in neurospheres, we used a mouse phospho-Receptor Tyrosine Kinase array (phospho-RTK array R&D, #ARY014) which was probed with OCAM KO and OCAM WT neurosphere extracts according to manufacturer’s recommendations. WB and arrays were quantified using ImageJ software.

Cells were treated with 5μg/ml anti-ErbB2 antibodies for 4h at 37°C. After washing, the cells were lysed in SDS lysis buffer and the cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against ErbB2, phospho-ErbB2-Tyr1221/1222, ErbB3, phospho-ErbB3-Tyr1289, AKT, phospho-AKT-Ser473, p44/42 MAPK, phospho-p44/42 MAPK-Thr202/Tyr204, SAPK/JNK, phospho-SAPK/JNK-Thr183/Tyr185, c-Jun, phospho-c-Jun-Ser63, PARP, Caspase-3, LC3A/B, Bak, Bax, Puma, Bid, Bim, Mcl-1, Bcl-xl, phospho-Bcl-2-Ser70, Bcl-2 (all from Cell Signaling Technology).

Cells were lysed directly in RIPA buffer and incubated on ice for 5 min. The lysate was then sonicated in a water bath sonicator (Diagenode Bioruptor) for 5 min, 30 s on/off and protein quantified using Pierce BCA Assay Kit (ThermoFisher, 23227). Lysates were supplemented with SDS-PAGE loading dye to a final concentration of 1x and boiled for 10 min. Equal amounts of protein were separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (GE life sciences, 1060002) using a Pierce Power Station (ThermoFisher). Membranes were blocked using Odyssey blocking buffer (Licor, 927–40000) and then incubated with antibodies against KLF5 (abcam, ab137676), Tubulin (Sigma-Aldrich, T9026), ERBB2 (ThermoFisher, MA5-14057), phospho-ERBB2 (Cell Signalling Technologies, 6942S), AKT (Cell Signalling Technologies, 2920S), phospho-AKT (Cell Signalling Technologies, 9106S), ERK1/2 (Cell Signalling Technologies, 4695S) or phospho-ERK1/2 (Cell Signalling Technologies, 9106S) overnight at 4°C. Membranes were incubated with IRDye secondary antibodies (Licor, 925–32212, 925–32213) and imaged using a Li-Cor Odyssey scanner.