Pepspots

Peptide spots (PepSPOTs, JPT Peptide Technologies GmbH) contain approximately 5–10 nmol peptide covalently bound to a cellulose-β-alanine membrane as described^{108 (link)}. The membrane was briefly rehydrated in PBS and blocked with PBS/2% BSA. The kinase reaction (30 °C for 30 min) was carried out in situ with recombinant 100 nM His-PAK4cat^{23 (link)} and 25 μM ATP (containing 1 μCi γ32P ATP) in 25 mM HEPES pH 7.3, 150 mM KCl, 10 mM MgCl₂, 2 mM dithiothreitol. The membrane was extensively washed in PBS/0.1% SDS buffer and subsequently exposed to X-ray film at −20 °C.

Identification of binding sites for recombinant SHP-2 and CSK in the caveolin-1 protein was performed using PepSPOTs (JPT Peptide Technologies GmbH, Berlin, Germany). Synthetic peptide sequences of caveolin-1 (1–85 and 1–28) were used in this study. The peptide library contained either 13-mer peptides that overlapped by 5 residues or 13-mer peptides that overlapped by 8 residues. Each spot carried approximately 5 nmol of peptide covalently bound to the cellulose-betaalanine-membrane (N-terminus: acetyl). Two identical membranes were incubated overnight with either recombinant GST-SHP-2 (rGST-SHP-2) or GST-CSK (rGST-CSK) respectively, and immunoblotting was performed to detect rGST-SHP-2/caveolin-1 peptide and rGST-CSK/caveolin-1 peptide interactions.

Overlapping peptides (15 mers overlapping by 4 amino acids) covering the entire p95HER2 extracellular domain and peptides with N-terminal truncations or AA substitutions for the shorter 20 mer epitope (peptide seq: “GVKPDLSYMPIWKFPDEEGA”) were generated and immobilized on a cellulose membrane (PepSpots, JPT Peptide Technologies GmbH, Berlin, Germany). The membrane was rinsed with methanol for 5 min at RT and washed three times for 3 min with TBST. The membrane was blocked by incubating with 5% nonfat skim milk (Bio-Rad, Oslo, Norway) for two hours at RT and then incubated with the Oslo-2 mAb (5 μg/mL) diluted in the same blocking solution for three hours at RT. After washing three times for 5 min in TBST, the membrane was incubated with an HRP-conjugated secondary antibody (horse-anti-mouse, 1.33 μg/mL) diluted in the same blocking solution for two hours at RT. The membrane was washed in TBST and incubated with SuperSignal West Pico chemiluminescent scent substrate (Thermo Fisher Scientific, Oslo, Norway) for 1 min. The membrane was washed repeatedly and gently with TBST and visualized by iBright FL1500 (Invitrogen, Waltham, MA, USA).