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. Author manuscript; available in PMC: 2010 Apr 6.
Published in final edited form as: J Immunol. 2009 Jan 1;182(1):547–553. doi: 10.4049/jimmunol.182.1.547

FIGURE 5.

FIGURE 5

Phospho-c-Raf (S338) levels are enhanced by GSK3 inhibition and are critical for the ability of GSK3-inactivation to augment phospho-ERK levels. A, Monocytes were pretreated with the GSK3 inhibitor SB216763 (10 µM) or LiCl (10 mM) followed by the addition of LPS (1 µg/ml) for 60 min. The levels of phospho-c-Raf (S338) in monocytes were determined by probing 20 µg of total cellular protein by Western blot. The immunoblot was reprobed for total p38 and the mean intensity ratio of phospho-Raf S338 to total p38 was calculated by densitometry. B, Monocytes were preincubated with the GSK3 inhibitor SB216763 (10 µM) or LiCl (10 mM) with or without the Raf-1 inhibitor (100 nM) for 90 min followed by the addition of LPS (1 µg/ml). Whole cell lysates were prepared 60 min post stimulation and 30 µg protein was probed by Western blot for phospho-MEK and phospho-ERK levels. The blot was reprobed for total β-actin levels to ensure equivalent loading. Data are representative of three experiments.