Anti pabp

IFA were performed as described previously [21 (link)]. We used anti-DCP1a, anti-eIF4E, anti-G3BP1 (Santa Cruz Biotechnology) and anti-PABP (Abcam) antibodies to detect P-bodies and stress granules. Fluorescence imaging was performed using a Leica TSC SP5 laser-scanning confocal microscope.

Total protein extracts were obtained from mock- or ASFV-infected cells and lysed in mRIPA. Quantification of total protein was done using a bicinchoninic acid (BCA) protein assay kit (Pierce) following the manufacturer′s protocol. For protein electrophoretic separation assays, 10 to 60 μg total protein was mixed with Laemmli buffer and electrophoresed in SDS-PAGE gels under reducing conditions. Following separation, proteins were transferred to Immobilon-P membranes (Millipore) and incubated with primary antibodies: anti-ASFV-DP or anti-p72 (1/1,000; generated in our laboratory), anti-actin (1/1,000; Santa Cruz Biotechnology), anti-tubulin (1/250; Sigma-Aldrich), anti-GFP (1/1,000; Roche), anti-PABP (1/200; Abcam), anti-RPS6 (1/1,000; 5G10; Cell Signaling), anti-RPL24 and anti-RPS6 (1/1,000; a kind gift from Anne Willis, MRC Toxicology Unit, Leicester, United Kingdom), or anti-RPL23a (1/500; a kind gift from Miguel Angel Rodriguez Gabriel, CBMSO, Madrid, Spain [71 (link)]), and an antibody able to recognize the inducible proteins of ASFV or anti-ASFV proteins (1/4,000; generated at CBMSO [72 (link)]). For detection, the chemiluminescent ECL Western blotting analysis system (Amersham Pharmacia) was used, following the manufacturer′s instructions.