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Fig. 1 siRNA mediated knockdown of robo4 and robo1. A shows RT-PCR gel for robo1 (R1), robo4 (R4) and actin (A) transcripts in the presence (+) of reverse transcriptase (RT) from HUVEC total RNA. The relative expression level of robo1 has percentile rank of 79 ± 4.5% and robo4 is slightly higher at 83.7 ± 5.7% in HUVECs [35,36]. Numbers on the bottom of gel represent lane numbers. B depicts RT-PCR for robo1, robo4 and actin genes in the presence (+) of reverse transcriptase (RT) from total RNA isolated from HUVECs transfected with increasing concentrations (bars) of robo4 siRNA. For robo1 gene: lanes 4 and 6, robo4 gene: lanes 10 and 12, and actin gene: lanes 16 and 18 represent 125 and 250 ng of siRNA respectively. Lanes 2, 8 and 14 are RNA isolated from untransfected HUVECs, and represent endogenous levels of each transcript. C indicates the ratios of robo1 and robo4 transcripts by real time PCR in control lacZ siRNA and robo4 siRNA transfected cells. D shows the RT-PCR for robo1, robo4, and actin transcripts in robo1 siRNA (lanes 1 and 2) or control lacZ (lanes 3 and 4) siRNA cells. E shows western blots for V5-human Robo4 and actin protein in 293 cell lysates from samples transfected with respective siRNA indicated by +. F shows western blot for endogenous Robo1 and Actin protein levels in control lacZ siRNA (Cont. siRNA) or robo1 (R1 siRNA) and untransfected (UT) 293 cells. Lanes 1 and 3, 2 and 4 are 250 and 500 ng of respective siRNAs indicated on the top of the gel. Lanes 15, 17 and 19 have spillover of excess actin product in -RT lanes.