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Figure 1


Fig. 1. Both Rac1 and Rac3 are endogenously expressed in N1E-115 cells, and are efficiently and specifically downregulated by shRNA. (A) Total RNA isolated from N1E-115 was used as template for cDNA synthesis. Primers specific for Rac1, Rac3 or GAPDH were used in semi-quantitative reverse-transcription PCR to show the relative expression levels of these genes. (B) Cells were transfected with pcDNA3.1 empty vector (ev), Rac3~FLAG or Rac1~HA, lysed and western blotted. Upper panel shows total Rac staining. Note that Rac3 antibody recognizes specifically Rac3 and not Rac1 (middle panel). beta-actin staining was used as loading control. (C) RT-PCR showing the expression levels of GAPDH (control; top panel), endogenous Rac3 (middle panel) and endogenous Rac1 (lower panel) in cells in which either shRac1 (lane 2, 3) or shRac3 (lane 4, 5) has been expressed. Control cells (lane 1) were transfected with siLuc. (D) Western blot showing depletion of Rac3 protein upon expression of shRac3, visualized by total Rac antibody (upper panel) and Rac3-specific antibody (middle panel). Beta actin staining is shown as loading control. (E) Western blot showing depletion of either Rac1 or Rac3 by shRac1 or shRac3, respectively. pSuper constructs, containing different sequences specifically targeting Rac1, Rac3 (e.g. shRac1-1, shRac3-1 and shRac3-2), or luciferase, were co-transfected with pcDNA3/Rac1~HA or pcDNA3/Rac3~FLAG constructs in HEK293 cells.





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