Luminol

Protein lysates were generated by mechanical and enzymatic treatment of cryo‐conserved human brain tumors, primary tumor cells and M/M as described previously 53. The protein concentration was determined according to the manufacturer’s protocol of the Micro BCA™ Protein Assay Kit (Thermo Scientific, Dreieich, Germany). The electrophoretic separation of the denatured proteins was performed on 12.5% SDS‐polyacrylamide gels followed by a blotting process as described previously 53. Blots were blocked in 1x Roti‐Block blocking buffer (Roth, Karlsruhe, Germany) and then incubated with the primary antibodies Iba1 (Wako, 016‐20001, dilution for WB 1:1000) and beta‐actin (Abcam, ab8227, dilution for WB 1:2500) as a loading control. Immunodetection was performed by HRP enzyme‐coupled secondary antibodies, which oxidize luminol (Santa Cruz Biotechnology, Heidelberg, Germany) resulting in a chemoluminescent reaction on X‐ray films (Super RX, Fujifilm Europe GmbH, Düsseldorf, Germany).

30 μg of protein were separated on polyacrylamide gels (Lonza), transferred to nitrocellulose membranes (Invitrogen) and blocked for 1 hour in blocking solution (2.5% milk powder (Biorad) in PBS containing 0.1% Tween-20 (PBS-T)). Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2^tyr1221/1222, p-EGFR^tyr1173, AKT, p-AKT^ser473, ERK, pERK^{thr202/tyr204}, eEF2, p-eEF2^thr56, mTOR, p-mTOR^ser2448, eEF2k, p-eEF2k^ser366 (Cell Signaling Technology), p-eEF2k^ser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich). Detection was performed using Luminol (SantaCruzBiotechnology) or ECL Advance (GE Healthcare).

Trypsin activated monomeric toxins were labeled with biotinyl-N-hydroxy-succinimide ester according to the manufacturer’s instructions (Amersham Biosciences). Binding of labeled toxins was analyzed by incubating 5 nM labeled toxin with 10 μg BBMV protein for 100 min at 25 °C in 100 μl binding buffer (PBS, 0.1% BSA, 0.1% Tween 20, pH 7.6). Non-specific binding was determined by measuring binding of 5 nM labeled toxin in the presence of 1000-fold molar excess of unlabeled toxin after 100 min. After incubation, the unbound toxin was removed by centrifugation for 10 min at 14,000 xg. The pellet containing BBMV and bound toxin was washed twice with 100 μl binding buffer, suspended in 10 μl of PBS pH 7.6, and 10 μl sample loading Laemmli buffer 2X (0.125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.01% bromophenol blue). Samples were boiled 3 min, loaded in 10% SDS-PAGE gels and electrotransferred to nitrocellulose membranes. Bound labeled toxin was identified by incubating with streptavidin-peroxidase conjugate (Millipore) (1:20000 dilution) for 1 h and developed with luminol (Santa Cruz Biotechnology Inc.). Binding assays were performed in triplicate.