Quantitative Western Blot Analysis

Tissue homogenates were standardized to DNA in SDS-PAGE sample buffer, as described 20 (link),21 (link), and separated by PAGE on a 4–12% BisTris gradient SDS gel and blotted onto polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA). Each gel had the same positive control (20 μg protein of 1% O₂ hypoxia-treated Lewis Lung whole-cell lysate) to normalize the signal between blots. Membranes were probed with primary antibodies against mouse HIF-1α (1/800; R&D Systems, Minneapolis, MN), GLUT-1 (1/20000; Abcam, Cambridge, UK), CA-IX (1/800, R&D Systems), or β-actin (1/10000, R&D Systems), and with secondary anti-goat or anti-mouse horseradish peroxidase-conjugated antibodies (DAKO, North Sydney, Australia). Membranes were developed using Amersham™ ECL Plus Western blotting detection system (GE Healthcare, Auckland, New Zealand), and protein bands were detected using a digital gel-doc system (Uvitec, Cambridge, UK). Multiple exposure times were used to ensure that the signal was not saturated, and measurements were taken in the linear range. Proteins bands were quantified using Alliance 2.7 software (Uvitec) and the protein of interest normalized against the positive control following confirmation of equal loading using β-actin.