Cells were lysed with RIPA buffer [1% NP40, 0.5% deoxycholate, 0.1% SDS, 150 mM sodium chloride, 50 mM Tris-HCl (pH 8.0)] supplemented with Halt protease inhibitor cocktail (Pierce, Rockford, IL), 5 mM EDTA, 5 mM sodium orthovanadate, 10 mM sodium fluoride and 1 mM β-glycerophosphate (Sigma). For preparation of lysates from cells cultured under hypoxic conditions, 100 μM CoCl
2 was added to the lysis buffer to prevent degradation of HIF-1α. Protein concentration was measured by Micro BCA Protein Assay Kit (Pierce). Lysates were mixed with Laemmli sample buffer (4X), boiled for 5 minutes at 100°C, and equal amounts of protein (50 μg per lane for a 10 comb gel) were separated by SDS-PAGE, followed by overnight wet-transfer to nitrocellulose at 4°C. For detection of CD26, samples were incubated at a lower temperature (37°C for 15 min). CD26 was detected at 220 kDa as a form of homodimer which increased detection sensitivity [
14]. We confirmed that the band detected at 220 kDa was depleted in a dose-dependent manner with anti-CD26 mouse monoclonal antibody 1F7 by immunoprecipitation (data not shown). After blocking with 5% nonfat milk in 0.1% Tween 20-TBS for 1 hour at room temperature, membranes were incubated with the appropriate primary antibodies in 1% blocking buffer for 2 hours at room temperature or overnight at 4°C [anti-CD26 (MI1004, Calbiochem, San Diego, CA or AF1180, R&D Systems, Minneapolis, MN), anti-Cdx2 (MU392A-UC, BioGenex, San Ramon, CA), anti-α-tubulin (T9026, Sigma, Saint Louis, MO), anti-c-Myc (9E10), anti-USF-1 (C-20), anti-HNF-1α (C-19) (sc-40, sc-229, and sc-6547, respectively, Santa Cruz, Santa Cruz, CA)], anti-HIF-1α (610958, BD Transduction Laboratories, San Jose, CA)], followed by incubation with the appropriate HRP-conjugated secondary antibodies (Pierce) for 1 hour at room temperature. Membranes were incubated with SuperSignal West Dura (Pierce) for 5 minutes, and visualized using a KODAK Image Station 2000R or 4000R (Carestream Health, New Haven, CT). Some membranes were processed using the Odyssey Infrared Imaging System by using the appropriate infrared-labeled secondary antibodies in Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE).