Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways
J. Clin. Invest. John Cijiang He, et al. 114:643 doi:10.1172/JCI21004 [Go to this article.]

Figure 7
Effects of PxxP and R₁₀₅R₁₀₆ motifs in Nef on Nef-induced MAPK1,2 and Stat3 phosphorylation and phenotypic changes in podocytes. Podocytes were infected with Nef constructs with a mutation in the PxxP or the R₁₀₅R₁₀₆ motif using a method similar to that described for WT Nef. (A) Western blot analysis was performed for phospho-Stat3, phospho-MAPK1,2, and β-actin. Densitometric data: phospho-Stat3/β-actin ratio: V = 0.18 ± 0.10, Nef = 0.62 ± 0.22, R₁₀₅R₁₀₆ = 0.39 ± 0.21, PxxP = 0.09 ± 0.02; phospho-MAPK1,2/β-actin ratio: V = 0.27 ± 0.10, Nef = 0.91 ± 0.23, R₁₀₅R₁₀₆ = 0.83 ± 0.12, PxxP = 0.43 ± 0.01; P < 0.05 when PxxP is compared with Nef, n = 4. (B) Western blot was also performed for cyclin E and β-actin. Densitometric data: cyclin E/β-actin ratio: V = 0.34 ± 0.22, Nef = 1.70 ± 0.26, R₁₀₅R₁₀₆ = 1.65 ± 1.11, PxxP = 0.81 ± 0.12, P < 0.01 when PxxP is compared with Nef, n = 4. (C) Northern blot analysis for synaptopodin, Nef, and GAPDH was performed as described. Nef expression was absent in cells infected with control vector. Densitometric data: synaptopodin/GAPDH ratio: Nef = 0.10 ± 0.06, V = 1.03 ± 0.21, PxxP = 0.41 ± 0.15, R₁₀₅R₁₀₆ = 0.48 ± 0.12, P < 0.01 when PxxP and R₁₀₅R₁₀₆ are compared with Nef, n = 3. (D) Cell proliferation was determined as described. After equal cell numbers (20,000) were plated per well, cell number was counted at day 3. Means of 3 experiments in triplicate are shown. *P < 0.001 as compared with Nef-infected cells.