RND3 Potentiates Proinflammatory Activation through NOTCH Signaling in Activated Macrophages

Figure 3

RND3 favors the translocation and expression of NOTCH1 in activated macrophages. (a) Western blot analysis of NOTCH1 expression in cytosol (left panel) and nucleus (right panel) in transiently transfected Raw 264.7 cells with control (Raw) or Rnd3 expression vector (Raw-RND3) activated with LPS (100 ng/ml) and IFN-γ (10 U/ml). ERK-2 expression was used as a loading reference for cytosolic and total protein extracts, and LAMIN-B for nuclear extracts. (b) Analysis by qPCR of Notch1mRNA expression in activated peritoneal murine macrophages transfected with Rnd3-specific siRNA and treated with LPS (100 ng/ml) and IFN-γ (10 U/ml) for 3 hr (c) Analysis of NOTCH1 protein expression in murine macrophages treated as previously described. (d) qPCR analysis of Notch1 in Rnd3^-/- bone marrow-derived macrophages (KORND3) and control macrophages (WT) activated 6 hr with LPS (100 ng/ml) and IFN-γ (10 U/ml). (e) qPCR analysis of Notch1 in Raw 264.7 cells stably transfected with a control (Raw) or Rnd3 expression vector (Raw-RND3) activated at 6 hr with LPS (100 ng/ml) and IFN-γ (10 U/ml). All qPCR data are referred to unstimulated macrophages set to 1. qPCR was performed in triplicate with riboprotein P0 as the internal control. Means ± SD of three independent experiments are shown. The Student unpaired t-test was used for statistical analyses between two groups at the level of , , , compared to control untreated cells.

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