Fig. 1. Subcellular location of overexpressed, endogenous and internalized S100A1. (A) Overexpressed S100A1 (AdS100A1; blue) is detected by an antibody that specifically reacts with S100A1. Western blot analysis comparing levels of S100A1 (upper) and cardiac actin (lower) expression in homogenates of Adcontrol and AdS100A1-transduced NVCMs. (B) Immunostaining of endogenous S100A1 (green) in untreated NVCMs reveals a fine granular network-like pattern (control). (C) Vesicular accumulation of internalized Rh-S100A1 protein (red) in the perinuclear region and in the cytosol of S100A1-treated cells. Western blot analysis comparing levels of S100A1 (upper) and cardiac actin (lower) in homogenates from S100A1-treated and control NVCMs. (D-F) AdS100A1-transduced cells were incubated for 1 hour with Rh-S100A1 24 hours post infection, and then immunolabeled with an antibody that specifically reacts with human S100A1. (D) The anti-human S100A1/Cy5-anti-rabbit antibody detects internalized Rh-S100A1 as well as overexpressed S100A1 protein (total exogenous S100A1). (E) Vesicular accumulation of internalized Rh-S100A1 protein in the perinuclear region and in the cytosol. (F) Overlay of both D and F shows that internalized Rh-S100A1 (violet) exhibits a distinct distribution compared with overexpressed S100A1 (blue). Bar, 10 µm.