Figure 8. Loss of p47^phox improves adriamycin-mediated activation of pro-fibrotic pathways.

(A) Equal amount of glomerular lysates (20 μg/lane) from 1 week adriamycin-treated WT, α1KO, p47^phoxKO, and DKO mice were analyzed by Western blot for levels of phosphorylated and total EGF receptor (EGFR). (B) pEGFR and EGFR bands were quantified by densitometry analysis and the levels of activated EGFR are expressed as pEGFR/EGFR ratio. Values are the mean ± SD of 3-4 mice/genotype. (C) Primary mesangial cells isolated from the mice indicated were cultured in serum free medium. Twenty-four hours later, cells were incubated with 2 μM dihydro-rhodamine and 2 hours later ROS generation was determined by FACS as described in the Methods. The level of ROS was expressed as a ratio of fluorescence intensity of cells with dihydro-rhodamine vs. that of cells without dihydro-rhodamine. Data represent the mean ± SD of 3 samples/genotypes. The same experiment was repeated twice with similar result. (D) Equal amount of cell lysates (20 μg/lane) from the serum starved cells indicated were analyzed by Western blot for the phosphorylated and total EGFR, activated and total Rac, phosphorylated and total ERK, as well as collagen IV (CIV) levels. (E) The collagen IV and β-actin bands were analyzed and expressed as described in Fig. 3. The values represent the mean ± SD of 3 independent experiments.