C digit digital scanner

Cell extracts were prepared by lysis with mild lysis buffer [NaCl 100 mM, Tris-HCl 10 mM pH 7.5, EDTA 1mM, NP40 0.5%, Protease inhibitor cocktail (Roche, 124 CH-4070 Basel Switzerland) and 30 μg of total protein were subjected to 10% SDS-PAGE and transferred to a nitrocellulose blotting membrane (GE Healthcare Boston, MA, USA). Membranes were incubated with an anti-eIF2α (Cell Signaling 32 Tozer Road Beverly, MA, USA), anti-p-eIF2α ser51 (Abcam Cambridge, CB2 0AX, UK) and anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Upon incubation with the corresponding HRP-conjugated secondary antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), membranes were revealed with a C-Digit digital scanner (Li-Cor Lincoln, NE, USA) using the PIERCE^(TM) ECL Western blotting substrate (Thermo, Waltham, MA, USA). Signal intensity was quantified by ImageJ (NIH).

Cells extracts from transfected cells were prepared by lysis with RIPA buffer and 20 μg of total protein were subjected to 10% SDS-PAGE and transferred to an Amersham Hybond™-P membrane (GE Healthcare). Membranes were incubated with an HIV-1 p24 monoclonal antibody diluted to 1/1000 (49 (link)), a rabbit anti-Flag antibody (Sigma-Aldrich) diluted to 1/1000 or and HRP-conjugated anti-actin antibody (Santa Cruz Biotechnologies) diluted to 1/750. Upon incubation with the corresponding HRP-conjugated secondary antibody (Santa Cruz Biotechnologies) diluted to 1/1000, membranes were revealed with the ECL substrate (Cyanagene) using a C-Digit digital scanner (Li-Cor).