Igf1 antibody

Total protein of CRC cell lines was extracted by RIPA lysis buffer (Solarbio, Beijing, China) and the concentration was measured using BCA Protein assay kit (Beyotime, China). Equal amounts of total protein (30 μg) were separated by 12% SDS–PAGE and transferred onto PVDF membranes. After blocking with 5% BSA in TBST buffer for 2 h, the membranes were incubated with IGF1 antibody (1:1000 dilution; 133542; abcam, Cambridge, UK), or β-actin (1:5000 dilution; 66009-1-lg; Proteintech, Chicago, IL, USA) overnight at 4°C. Afterwards, the membranes were incubated with a corresponding HRP-labelled secondary antibody (1:5000, Lablead, Beijing, China) for 1 h at room temperature. Finally, the blots were visualized using an enhanced chemiluminescent kit (Beyotime, China). Quantification of the individual protein bands was performed by densitometry using ImageJ software.

Excised tumours, treated as indicated, were stained with IGF-1 antibody (1 : 125; Abcam, Cambridge, UK) and detected using IHC Detection Kit (Invitrogen) (further details in Supplementary Information). Scoring was carried out on 5 fields of view at least 20 μm apart (2 sections per slide, two slides per tumour). Staining intensity was scored as negative (0), light (+ 1), medium (++ 2) or dark (+++ 3). Staining extent (i.e., the percentage of cells with cytoplasmic immunoreactivity) was scored as: negative (0), ⩽10% (1), 11–25% (2), 26–50% (3), 51–75% (4) and ⩾76% (5). Extent and intensity scores were added together to provide a score range of 0–8 (Braconi et al, 2008 (link)). Scores were averaged for three tumours per treatment group.