Journal: Nucleic Acids Research

Article Title: Modulation of HIV-1 gene expression by binding of a ULM motif in the Rev protein to UHM-containing splicing factors

doi: 10.1093/nar/gkz185

Figure Lengend Snippet: W45-dependent effect of Rev on the biogenesis of RRE-containing mRNA in HeLa cells. ( A ) Schematic representation of pDM128. The intronic sequence is represented as a dashed line, SD4 – splice donor 4, SA7 – spliced acceptor 7. The location of qPCR amplicons detecting either spliced or unspliced mRNA is shown. (B, C) HeLa cells were transiently transfected with pDM128 and Rev WT or W45A (+ pRLSV40 to control for transfection efficiency and pBSIISK+ as plasmid DNA for dilution). Total RNA was isolated 48 hours after transfection, reverse-transcribed with an oligo(dT) primer. ( B ) The relative abundance of spliced to unspliced poly(A)+ RNA was assessed by qPCR (left graph). ( C ) In order to look at the levels of spliced and unspliced mRNAs separately instead of as a ratio, the same qPCR data were plotted comparing changes in the levels of spliced and unspliced mRNA species in cells transfected with RevW45A versus Rev WT (right graph). ( D ) HeLa cells were transiently transfected with GFP-tagged Rev WT or W45A and assayed 48 hours after transfection. Extracts from HeLa cells were incubated with or without RNase A and subjected to immunoprecipitation with α-GFP antibody. 1% of input and 50% of the immunoprecipitate was analyzed by Western blot, using α-GFP and α-U2AF65 antibodies. The experiments were performed three times, a representative gel is shown.

Article Snippet: The following primary antibodies at 1:1000 dilutions were used: mouse mAb α-U2AF (MC3, kind gift of Karla Neugebauer, MPI-CBG), rabbit pAb α-GFP (Cell Signaling), rabbit pAb α-SPF45 (kind gift of Juan Valcarcel, CRG).

Techniques: Sequencing, Transfection, Plasmid Preparation, Isolation, Incubation, Immunoprecipitation, Western Blot