Advanblock fluor

Cell lysates were prepared in TXLB lysis buffer [50 mM Hepes, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM EDTA, 50 mM NaF, 10 mM Na₃VO₄, and protease inhibitor cocktail (cOmplete Mini, EDTA-free, Roche)], and volumes were adjusted according to protein concentration measurements (DC protein assay kit, Bio-Rad, 5000111). Separation was performed by gel electrophoresis (Mini-PROTEAN TGX Precast Gels 4-20%, Bio-Rad, 4561096), before transferring onto a nitrocellulose membrane (Trans-Blot Turbo Transfer System, Bio-Rad) and blocking with AdvanBlock-Fluor (Advansta, R-03729-E10). Primary antibodies in AdvanBlock-Fluor were incubated overnight at 4°C; IGFBP2 (R&D Systems, AF674), IGF-II (Millipore, 05-166-MI), GAPDH (Hytest, 5G4MAB6C5), and GFP (Thermo Fisher Scientific, A11122). Membranes were washed between primary and secondary antibody treatments with Tris-buffered saline with 0.1% Tween 20 (TBST). IRDye secondary antibodies (1:5000 diluted in TBST; LI-COR) were incubated for at least 1 hour at RT, before detection on an Odyssey fluorescence imager CLx (LI-COR). Densitometry analysis was performed in FIJI (NIH) by normalizing the signal to GAPDH, which was used as a loading control.