Ab60933

The proteins were transferred to a nitrocellulose membrane (66485, Pall Corporation, USA) using Trans Blot Turbo (1704270, Bio-Rad). Membranes were blocked in 10% non-fat milk in Tris buffered saline with Tween (TBST) at room temperature for 1 h and probed with primary antibodies diluted 1:1000-1:10 000 in 5% milk in TBST at 4 °C overnight. The primary antibodies were as follows: anti-drebrin (GTX12350, GeneTex; ab60933, Abcam), anti-GFP (GTX26673, Genetex; ab32146, Abcam), anti-AChR-α1, α3, α5 (838301, BioLegend), anti-rapsyn (ab156002, Abcam), anti-GAPDH (sc-25778, Santa Cruz) and anti-tubulin (ab18251, Abcam). After washing with TBST, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase [anti-rabbit HRP (111-035-144, Jackson Immuno Research, USA), anti-mouse HRP (7076, Cell Signaling, USA), anti-rat HRP (ADI-SAB-200-J, Enzo Life Sciences), and anti-goat HRP (sc-2020, Santa Cruz)]. Proteins were detected with Femto chemiluminiscent substrate (34095, ThermoFisher Scientific) and developed on X-ray films (771468, Carestream, USA).

DBN1 protein expression was assessed in a well-characterised series of luminal primary invasive breast cancer patients (n = 436), with long-term follow-up. Patients presented at the Nottingham City Hospital (1989–2006), as previously described [34 (link)]. The characteristics of this cohort are summarised in Table S2.
The specificity of DBN1 antibody (Ab60933, Abcam, UK) was validated prior to the staining by Western blotting using MCF7 human breast cancer cell lysate (American Type Culture Collection; Rockville, MD, USA), as previously described [34 (link)]. The specificity of the DBN1 antibody was observed with a single band at the predicted size of approximately 100 kDa (Figure S7). Immunohistochemistry (IHC) was used to assess DBN1 protein expression on 4-μm tissue microarray sections using Novolink polymer detection system (RE7150-K, Leica Biosystems, UK), as previously described [34 (link)] using the DBN1 antibody at a 1:1000 dilution. Evaluation of cytoplasmic staining for DBN1 in invasive tumours cells was based on a semi-quantitative assessment of invasive tumour cells using a modified histochemical score (H-score) [35 (link)]. Tissue microarray cores were only assessed if the invasive tumour burden was > 15%.