Figure 10. Knockdown of the Rho guanine exchange factor (GEF) p115 prevents KapB-induced modification of p-body dynamics.

A-B) HUVECs were sequentially transduced with two populations of recombinant viruses: firstly, puromycin-resistant viruses that express KapB or the empty vector; and secondly, GFP-expressing lentiviruses that express short hairpin RNAs (shRNAs) against a two different Rho guanine exchange factors (GEFs; p115 [numbered −3, −4, and −9], H1 [numbered −1, −2, and −7], or the non-specific (NS) shRNA control. Positive transductants were selected by puromycin treatment (1^st step) and positive GFP-expression, to mark shRNA-expressing cells (2^nd step). After seeding cells on coverslips, and a one-hour treatment in basal media the following day, cells were stained with the following primary antibodies: mouse anti-hedls (to stain p-bodies, false-colored blue) and rabbit anti-KapB (to stain KapB, red). Representative IF images are shown in A. To quantify p-body disruption, the number of cells expressing the transgene of interest (red) and the shRNA construct (green) that retained normal p-bodies was counted as for Fig. 4 and shown in B (n = 3 independent experiments). Scale bar  = 10 µm.