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. Author manuscript; available in PMC: 2008 May 30.
Published in final edited form as: Cell. 2007 Nov 30;131(5):980–993. doi: 10.1016/j.cell.2007.09.027

Figure 4. MAPK and GSK3 Phosphorylations Target Smad1 and Total Polyubiquitinated Proteins to the Centrosome in Cos7 Cells.

Figure 4

(A–C) pSmad1Cter accumulates in the centrosome (arrows) in response to proteasomal inhibition by Lactacystin (24%, n=323).

(D) The proteasomal subunit S20α5 normally accumulates in the centrosome in Cos7 cells (arrows).

(E and F) Nuclear and centrosomal localization of pSmad1MAPK.

(G–I) pSmad1GSK3- A (green) colocalizes with the centrosome marker γ-Tubulin (red). Note that in panels E through I BMP4 was not added, but the medium contained 10% fetal calf serum, which provides growth factors such as BMP and FGF.

(J and K) Specificity of pSmad1MAPK and pSmad1GSK3-A staining: phospho-peptide competition and λ-phosphatase sensitivity.

(L) pSmad1GSK3 accumulates in the centrosome 8 hrs after Lactacystin (50 μM) treatment (a 25-fold increase in volume).

(M–O) Treatment of Cos7 cells with Lactacystin induces accumulation of total cellular polyubiquitinylated proteins in the centrosomal region marked by Pericentrin in red. In one cell a ring of pericentrosomal staining is seen.