rabbit α eif4e  (Cell Signaling Technology Inc)

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Measles vaccine strains for virotherapy of non-small cell lung carcinoma

doi: 10.1097/JTO.0000000000000214

Figure Lengend Snippet: A) H2009, A549, and H838 cell lysates were assayed by immunoblot before and 48 hours after infection with MV-GFP for key proteins involved in cap-dependent and independent translation initiation. B) Beas2B expressing empty vector (Beas2B-V) and Beas2B expressing eIF4E (Beas2B-E) were infected with MV-CEA at indicated MOI and assayed for cell viability 72 hours after infection. 5’ cap-affinity assay of untreated Beas2B-V and Beas2B-E are shown. Relative increases in eIF4G binding in the cap-affinity assay correspond to enhanced eIF4F cap-complex formation. C) H2009 and H522 NSCLC cells were treated with MV-CEA (MOI=0.01), rapamycin (Rap), or the combination and assayed for cell viability 72 hours after infection. Supernatants were collected from cells and assayed for CEA production as a measure of viral replication. Error bars indicate standard deviation of the mean. * indicates statistical significance. D) H2009 and H522 cells were treated with MV-CEA (MOI=0.01), 4EGI-1 (15μM), or the combination and cell viability assayed 72 hours after infection. E) NSCLC cell lines were treated with MV-GFP at MOI of 0.25 either alone or in combination with rapamycin. Representative fluorescence micrographs are shown.

Article Snippet: The primary antibodies employed were rabbit α-eIF4E, rabbit α-4E-BP1, rabbit α-PARP, α-eIF2α, α-phospho-eIF2α(Ser 51 ), α-PKR, α-phospho-PKR, all from Cell Signaling at a 1:1000 dilution, mouse α-b-actin (Sigma) at a 1:10,000 dilution and rabbit α-eIF4GI (kindly provided by Nahum Sonenberg, McGill University Montreal, Quebec, Canada) at a 1:2500 dilution.

Techniques: Western Blot, Infection, Expressing, Plasmid Preparation, Binding Assay, Standard Deviation, Fluorescence