Knockdown of exogenous and endogenous EFNA1. For exogenous knockdown of EFNA1, HeLa cells were transiently transfected with EFNA1 cDNA and with either empty vector control, EFNA1 shRNA#1, or EFNA1 shRNA#2 plasmids. A) Western blot of 3 independent experiments showing the typical level of knockdown of exogenous EFNA1 (top). Blot was stripped and reprobed with an anti-β-actin antibody to show equal loading between lanes. B) Quantification of knockdown showing the average expression of EFNA1 relative to the amount of β-actin +/- the standard deviation. C) Endogenous EFNA1 knockdown. HeLa cells were transiently transfected with either empty vector control, or EFNA1 shRNAi#1. Shown are duplicate transfections. Western blotting using an anti-EFNA1 antibody showed a knockdown of endogenous EFNA1 in both conditioned medium and lysates (arrow). Below is a western blot using anti-Grb2 which was used to confirm equal loading of lysate lanes. * shows two cross-reacting bands which were used to confirm equal loading of conditioned medium lanes.