FIGURE 2:

LPS up-regulates cellular level of Lyn expressed in RAW264 cells. (A) Lyn-GFP constructs studied, with sites of mutations indicated. (B) Cellular distribution of indicated Lyn-GFP constructs. Arrows indicate submembraneous Lyn. Bar, 10 μm. (C, D) Lyn protein level in transfected RAW264 cells, as analyzed by immunoblotting and densitometry. Cells were cultured in 10% FBS (C) or shifted for 2 h to 2% FBS (D) before a 30-min stimulation with 100 ng/ml LPS. The level of Lyn variants in cell lysates was examined by immunoblotting with anti-Lyn antibody, normalized against actin content, and expressed in histograms relative to that of Lyn KD in unstimulated cells. Results are mean ± SD of three or four experiments. *Significantly different from unstimulated cells at p ≤ 0.05. (E) LPS-induced activation of Lyn revealed by immunoprecipitation of Lyn-GFP constructs and analysis of immunoprecipitates with antibodies directed against phosphotyrosine 397 (p-Tyr307) or phosphotyrosine 508 of Lyn (p-Tyr508). Efficiency of immunoprecipitation determined by blotting with anti-GFP antibody.