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. 2020 Apr 23;11:1943. doi: 10.1038/s41467-020-15732-9

Fig. 3. SNCA modulates the activation of ERK1/2, Akt and p38 in vitro.

Fig. 3

a, b HK-2 cells were incubated separately with either serum-free medium or 2 ng/ml TGF-β1 for 60 min. Cell lysates were immunoblotted with antibodies against pERK1/2, p-p38, pAkt, total ERK1/2, and tubulin. Representative Western blots (a) and quantitative densitometric analysis (b) show levels of phosphorylated ERK1/2, p38, and Akt in HK-2. Data were normalized to total ERK1/2 (b) and presented as mean ± SEM of at least n = 3 independent experiments. c–e Effect of MAPK inhibitors (U0126 and SB203580) and PI3K-Akt inhibitor (LY294002) on TGF-β1-induced decrease in SNCA expression (c, d), as well as SNCA knockdown-induced increase of vimentin in HK-2 (e). HK-2 were pretreated for 1 h with UO126, (UO; 10 µM/L), SB203580 (SB; 20 µM/L) or LY294002 (LY; 10 µM/L), after which cells were incubated either with serum-free medium or TGF-β1 (2 ng/ml) for 72 h. Cell lysates were immunoblotted with antibodies against SNCA (c, e), vimentin (e) and tubulin. Representative Western blots (c, e) and quantitative densitometric analysis (d) show levels of SNCA (c, e) and vimentin (e) in HK-2. Data were normalized to tubulin (d) and presented as mean ± SEM of at least n = 3 independent experiments. f The confocal Z stack shows colocalization of p38 with SNCA in the cytoplasm of HK-2. HK-2 cells were grown under normal conditions and were stained using anti-SNCA (red) and anti-p38 antibody (green). Nuclei were labeled by Hoechst (blue). The lower and right panels in the confocal Z stack show a vertical cross section (yellow lines) through the cells. Arrow, SNCA colocalized with p38 MAPK in the cytoplasm. Scale bar represents 10 µm. g Immunoprecipitation of SNCA with p38. HEK293T cells were co-transfected with p38-HA and SNCA-flag. After crosslinking with DSP, p38 was immunoprecipitated from the cell lysates using antibodies against HA, as explained in Supplementary Methods. Lysates and immunoprecipitates were probed on Western blots with antibodies against flag and HA. *p < 0.05, **p < 0.01, ***p < 0.001. The p-value by two-way ANOVA. C—control. Source data are provided as a Source Data file.