Irdye polyclonal secondary antibodies

To prepare protein lysates, cells were treated with M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA) supplemented with HALT Protease Inhibitor Cocktail (Thermo Scientific) and Phosphatase Inhibitor Cocktail (Thermo Scientific). Fifteen micrograms of total protein were electrophoresed per well on a SDS-polyacrylamide gel and transferred onto Immobilon-FL PVDF membranes (Millipore, Billerica, MA, USA). Membranes were then incubated with primary antibodies (Additional file 6: Table S4) and probed with a mixture of IRDye polyclonal secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Images were read with an Odyssey infrared imaging system (LI-COR Biosciences) and the average density of each band was measured with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells were treated with M-PER^® Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA), HALT Protease Inhibitor Cocktail (Thermo Scientific), and Phosphatase Inhibitor Cocktail (Thermo Scientific) to extract total protein. Approximately, 15–20 µg of total protein were electrophoresed per well on an 8–10% SDS-polyacrylamide gel and transferred onto Immobilon-FL PVDF membranes (Millipore, Billerica, MA, USA). Blots were incubated with the HIF-1α primary antibody (H1alpha 67): sc-53546 (Santa Cruz Biotechnology, CA, USA) at 1:500 dilution and monoclonal GAPDH antibody (MAB374, from Millipore, Billerica, MA, USA) at 1:10,000 dilutions overnight. After blocking with primary antibodies, membranes were washed and then probed with a mixture of IRDye polyclonal secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Images were read with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Protein samples were isolated from an independent series of paired specimens of polypoid (n=10) and ulcerative (n=10) tumors and normal (non-cancerous) mucosa using PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea). An equal amount of protein in each sample was quantified using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Cellular proteins were separated by 15% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked in 5% milk solution for 30 min at room temperature. The membranes were then incubated with rabbit monoclonal anti-S100P at a 1:1,000 dilution (clone EPR6143; Epitomics, Burlingame, CA, USA) at 4°C overnight, followed by incubation with IRDye polyclonal secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Signals were detected using the ECL Detection system (Bio-Rad) as per the manufacturer’s instructions. Mouse monoclonal anti-cytokeratin AE1/AE3 antibody (cat. no. MAB3412) and mouse monoclonal anti-GAPDH antibody (cat. no. CB1001, both 1:1,000 dilution; Millipore, Billerica, MA, USA) were used as the controls for normalization. Densitometric analysis of the bands compared with the density of AE1/AE3 and GAPDH was performed using ImageJ software, as previously described (19 (link)).