Ez ecl hrp chemiluminescence detection kit

Western blotting was performed as describe in^{15 (link)}. Protein lysates were prepared from purified cDCs using RIPA buffer with phosphatase and protein inhibitors (Pierce). Proteins were separated with SDS-PAGE and transferred to Immobilon™ PVDF membrane (Millipore). Membranes were blocked for 1 h at room temperature in PBST containing 5% (w/v) milk, incubated overnight at 4 °C with anti-Hif1α antibody (Cell signalling) and subsequently with HRP-conjugated secondary antibody (Amersham Bioscience). Blotted proteins were detected using an EZ-ECL™ HRP chemiluminescence detection kit (Biological Industries) and exposed on to Hyperfilm™ photo film (Amersham).

BHK-21 cells were transfected with RNA transcripts from pGFP-PAC or pLL-GFP-PAC, cell extracts prepared at 1, 2, 4, 6, 8, 10 and 24 h post-transfection using RIPA lysis buffer (150 mM NaCl, 50 mM Tris–HCl [pH 7.4], 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS). Samples were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane (Life Technologies) and blocked for 1 h in 5% PBS-T (PBS, 0.1% Tween 20, supplemented with 5% non-fat milk). The membrane was probed with rabbit anti-eIF4G (1:1000: kind gift of Lisa Roberts) or mouse anti-tubulin (Invitrogen, 1:2000) overnight at 4 °C. The membrane was then washed 3× in PBS-T, then probed with peroxidase conjugated anti-rabbit (Invitrogen, 1:2000) or anti-mouse (Invitrogen, 1:2000) antibodies in 5% PBS-T for 1 h at room temperature. Membranes were washed 3× in PBS-T and antibody binding detected using an EZ-ECL HRP chemiluminescence detection kit Biological Industries, Kibbutz Beit-Haemek, Israel).