Recombinant human hb egf

PD98059 and Ly294002 were purchased from Cell Signaling Technology (Danvers, MA). Recombinant human HB-EGF was purchased from R&D Systems (Minneapolis, MN). The following primary antibodies were used: anti-GPC1 antibody from Atlas antibodies (Stockholm, Sweden); anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-p70S6K (Thr389), anti-p70S6K, anti-phospho-p44/42 (Thr202/Tyr204), anti-p44/42, anti-Bak, anti-Bim, anti-Bcl-w and anti-phospho-EGFR (Tyr1068) from Cell Signaling Technology; anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-EGFR from BD Transduction Laboratories (San Jose, CA).

Human podocytes were cultured as previously described^{14 (link)}. Briefly, podocytes were grown under permissive conditions at 33 °C temperature in media containing 0.01 mg/ml recombinant human insulin, 0.0055 mg/ml human transferrin. We developed stable podocyte cell lines expressing ZFYVE28 by transfecting the cells using Lipofectamine 2000 (Thermo fisher scientific). The cells were transfected with pRP(Exp)-CMV > hZFVE28(ORF0226721):T2A:Puro/3xFLAG (VectorBoulder.com) or with an empty vector. Clones were obtained by limited dilution and expanded under puromycin selection. The over-expression of ZFYVE28 in different clones was confirmed by western blotting. The cells were treated with 100 ng/ml of recombinant human HB-EGF (R&D systems, cat: 259-HE) at different time points.

All tissue culture components and solutions were purchased from Gibco BRL (Paisley, UK). Recombinant human bFGF and recombinant human HB-EGF were purchased from R&D Systems (Abingdon, UK). AG1478, LY294002, PD98059, and SP600125 were obtained from Calbiochem (Bad Soden, Germany). SB203580 was from Tocris (Ellisville, MO). Human-specific small interfering RNA (siRNA) against nuclear factor of activated T cell 5 (NFAT5) and nontargeted control siRNA were obtained from Qiagen (Hilden, Germany). All other agents used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. The following antibodies were used: a neutralizing goat anti-bFGF (R&D Systems), a neutralizing goat anti-HB-EGF (R&D Systems), a neutralizing rabbit anti-transforming growth factor (TGF)-β (pan specific; R&D Systems), a rabbit anti-human NFAT5 (1:200; Santa Cruz Biotechnology, Dallas TX), a rabbit anti-human β-actin (1:1000; Cell Signaling, Frankfurt/M., Germany), and anti-rabbit IgG conjugated with alkaline phosphatase (1:2000; Cell Signaling).