FIG 1.

Golgi UDPase and LALP70 enhance E4orf4-induced cell death. Plasmids expressing UDPase-Myc, LALP70-Myc, or an empty vector were transfected into 293T cells with a plasmid expressing E4orf4 or with the corresponding empty vector. (A) Cell extracts were subjected to immunoprecipitation (IP) with antibodies to the Myc tag, and the presence of E4orf4 and LALP70-Myc proteins in the immune complexes and in input lysates was determined by a Western blot. Alpha-tubulin served as a loading control. The amount of proteins in the input represents 10% of the amount of proteins used for immunoprecipitation. (B) Cells in duplicate plates were stained 24 h after transfection with antibodies to E4orf4 and the Myc tag and with 4′,6-diamidino-2-phenylindole (DAPI). Nuclei with apoptotic morphology were counted in the double-transfected cell population expressing both E4orf4 and UDPase-Myc and in single-transfected cells expressing either E4orf4 or UDPase-Myc, and the percentage of cell death was determined. Cell death induced by E4orf4 alone was defined as 100%, and relative cell death was calculated for the other samples. Two independent experiments, each with duplicates, were carried out. Error bars represent the pooled standard deviation, and statistical significance was determined using a one-tailed t test (*, P = 0.043; **, P = 0.04). (C) Proteins extracted from a parallel set of E4orf4-transfected plates, as described for panel B, were analyzed by Western blotting using antibodies to E4orf4 and to alpha-tubulin. (D) Representative photographs of cells containing the indicated plasmids were taken 24 h after transfection.