pathog 5 e1000287 ikegami et al 2005 j virol 79 12106 12111 western blot analysis western blot analysis was performed as described previously ikegami et al 2009 plos pathog 5 e1000287 the membranes were incubated with anti pkr antibody b 10 santa cruz biotech anti eif2 alpha antibody cell signaling tech anti phospho eif2 alpha ser51 antibody cell signaling tech anti rvfv mouse polyclonal antibody a kind gift from dr r b tesh utmb anti dykddddk tag antibody cell signaling tech or anti actin goat polyclonal antibody i 19 santa cruz biotech overnight at 4 u00b0 c and with secondary antibodies for 1 hr at room temperature analysis of viral growth mef cells were infected with viruses at a multiplicity of infection moi of 0 01 at 37 u00b0 c for 1 hr culture supernatants were harvested at 72 hpi and the virus titer was measured by plaque assay the growth curves were shown as mean u2212 standard deviation from three independent experiments analysis of host protein synthesis mef cells were infected with viruses at a moi of 3 at 37 u00b0 c for 60 min at 16 5 h pi cells were incubated in mem containing 1 dialyzed fbs and 4 mm l glutamine for 30 min and then incubated in medium containing 100 u03bcci ml of 35 s methionine for 30 min cell lysates were separated by 10 polyacrylamide gel electrophoresis and analyzed by autoradiography the same lysates were also analyzed by coomasie blue staining to compare the amounts of loaded proteins analysis of general transcription mef cells were mock infected or infected.