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. 2018 Nov 2;132(21):2299–2322. doi: 10.1042/CS20180563

Figure 2. Inhibition of autophagy suppresses the TGF-β/Smad, Wnt/β-catenin, EGFR/ERK1/2 signaling pathway in renal interstitial fibroblasts.

Figure 2

Cultured NRK-49F cells were starved for 24 h and then exposed to 800 μM of uric acid for 36 h in the absence or presence of 3-MA (0–10 mM). Cell lysates were subjected to immunoblot analysis using antibodies to TGF-βRІ, p-Smad3, Smad3, or β-Actin (A). Expression levels of TGF-βRІ were quantitated by densitometry and normalized with β-Actin; expression levels of p-Smad3 and Smad3 were calculated by densitometry and the ratio between p-Smad3 and Smad3 was determined (B). Cell lysates were prepared and subjected to immunoblot analysis with antibodies to Wnt1, β-catenin, and GAPDH (C). Expression levels of Wnt1 were quantitated by densitometry and normalized with GAPDH and expression levels of β-catenin were quantitated by densitometry and normalized with GAPDH (D). Cell lysates were prepared and subjected to immunoblot analysis with antibodies to p-EGFR, EGFR, p-ERK1/2, ERK1/2, and GAPDH (E). Expression levels of p-EGFR and EGFR were calculated by densitometry and the ratio between p-EGFR and EGFR was determined (F). Expression levels of p-ERK1/2 and ERK1/2 were calculated by densitometry and the ratio between p-ERK1/2 and ERK1/2 was determined (F). Data are represented as the mean ± S.E.M. Means with different superscript letters are significantly different from one another (P<0.05).