Journal: Journal of Virology
Article Title: Stress-Inducible Alternative Translation Initiation of Human Cytomegalovirus Latency Protein pUL138 ▿
Figure Lengend Snippet: UL138 transcripts are polycistronic. (A) HEK-293 cells were transfected with cDNAs representing the 3.6-, 2.7- and 1.4-kb UL138 transcripts or constructs encoding individual myc-tagged ORFs and analyzed by immunoblotting using polyclonal rabbit antisera specific to pUL133, pUL135, pUL136, and pUL138 or a monoclonal antibody specific to tubulin as a loading control. (B) The intensities of the pUL138 band were quantitated with a Li-Cor Odyssey imaging system and are represented graphically. (C) RNA isolated from lysates described in panel A was analyzed by qRT-PCR. Inverse crossing points (1/Ct) were determined using the Roche LightCycler 480 software with the second derivative max method (graphed). Fold change compares amplifications of the 5′ and 3′ ends of experimental cDNAs isolated from transfected cells to cDNA derived from full-length iv-RNA. Transcripts with fold changes ≤2 compared to iv-RNA were considered to be full length.
Article Snippet: Quantitative reverse transcription PCR (qRT-PCR) was performed with the LightCycler 480 Probes Master (Roche) according to the manufacturer's instructions along with Universal Probe Library (Roche) probes and primers specific for UL133 (forward, 5′-TTTCTGGACCTGGCATCG-3′; reverse, 5′-TACGCGCAGAGGAAAATCAT-3′; probe number 114, 5′-CTGTGGTC-3′), UL135 (forward, 5′-GCGGTGTACGTCGCTCTAC-3′; reverse, 5′-GGAAACTCGGGTTTATCTATCG-3′; probe number 55, 5′-GGCAGACCATCCTCTCCTATG-3′), UL136 (forward, 5′-GCGGCTGTCATTATCCTGAG-3′; reverse, 5′-TAGTACATGGCCCCGTTCA-3′; probe number 152, 5′-TCGCCGTC-3′), UL138 (forward, 5′-GGTTCATCGTCTTCGTCGTC-3′; reverse, 5′-CACGGGTTTCAACAGATCG-3′; probe number 126, 5′-TCTCTGGT-3′), and firefly luciferase (FL) (forward, 5′-TGAGTACTTCGAAATGTCCGTTC-3′; reverse, 5′-GTATTCAGCCCATATCGTTTCAT-3′; probe number 29, 5′-GAAGACGG-3′) and the prevalidated UPL human beta-actin gene assay (Roche).
Techniques: Transfection, Construct, Imaging, Isolation, Quantitative RT-PCR, Software, Derivative Assay