Peroxidase conjugated secondary antibody

Total cellular extracts and soluble nuclear extracts from HRPTECs were prepared as described previously^{57 (link),58 (link)}. Western blotting was carried out using 3–8% Novex NuPAGE Tris-acetate gels (Invitrogen) for HIF-1α and nucleoporin p62 or 4–12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen) for p-AMPKα (Th172), AMPK and α-actinin under reducing conditions. After proteins were transferred onto a Hybond-P PVDF membrane (Amersham Biosciences Co., Piscataway, NJ, USA), the membranes were incubated with the primary antibodies (dilution 1:1000), incubated with a peroxidase-conjugated secondary antibody (dilution 1:50000) (Amersham), and visualized with an enhanced chemiluminescence (ECL) system (Amersham). Selected blots were washed and reprobed with an antibody against nucleoporin p62 for nuclear protein extracts and α-actinin for total cellular extracts to control for small variations in protein loading and transfer. Images were processed using ImageJ (U. S. National Institutes of Health, Bethesda, MD, USA) for densitometric analysis. Signal intensities in the control lanes were arbitrarily assigned a value of 1.00.

Cells were harvested and washed twice with PBS. Cell lysis was performed on ice for 25 min, in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitory cocktail (Roche). Insoluble material was pelleted by centrifugation at 16,000× g at 4°C for 3 min. Protein concentrations were determined using the Bradford assay (Bio-Rad). Thirty micrograms extract was mixed with 4× Laemmli buffer (40% glycerol, 240 mM Tris/HCl, pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol), denatured at 96ºC for 5 minutes, separated by SDS-PAGE, and transferred to nitrocellulose membranes (PROTRAN-Whatman, Schleicher&Schuell). The membranes were blocked with 5% non-fat dry milk in TBS-T for 60 min, incubated with primary antibodies overnight at 4°C, washed three times with TBS-T for 10 min, incubated with the peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences) in TBS-T with 5% non-fat dry milk for 60 min, and washed three times with TBST for 10 min. Immunoreactive proteins were detected using Supersignal West Dura HRP Detection kits (Pierce). The primary antibodies used were: p16^Ink4a (Santa Cruz); p19^Arf (Ab80 Abcam); p15^Ink4b (Santa Cruz); β-catenin (clone 14, BD Biosciences); p53 (sc-6243 Santa Cruz); p21 (BD Pharmigen); c-Myc (sc-764 Santa Cruz); β-actin (ab8226, abcam).

Protein samples were resolved on SDS–polyacrylamide gels and transferred to Hybond-P membranes (Amersham, Pittsburgh, PA). Membranes were blocked for 1 h in TBS, 0.1% Tween-20 and 5% BSA, followed by an overnight incubation with primary antibody diluted in the same buffer. After washing with 0.1% Tween in TBS, the membranes were incubated with peroxidase-conjugated secondary antibody (Amersham) for 1 h, and then washed and developed using the ECL chemiluminescent detection system (Amersham). A human specific rabbit anti-hSOD1 (clone EPR1726, Epitomics, CA) was used to probe for hSOD1. Densitometric analyses were performed using the NIH Image software and normalized against the signal obtained by re-probing the membranes with anti-actin (clone AC-15, Sigma-Aldrich).