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. 2021 Apr 8;11:7781. doi: 10.1038/s41598-021-87435-0

Figure 4.

Figure 4

The neuroprotective effect of GSB-106 is mediated through the activation of MAPK/Erk and PI3K/Akt pathways. (A) SH-SY5Y cells were incubated in serum-free culture medium (“0% FBS”) with or without BDNF (28.6 ng/ml, ~ 1 nmol) or GSB-106 (100 nmol) for 10, 30 or 180 min. After incubation, cells were collected, and protein extracts were subjected to polyacrylamide gel electrophoresis and transferred for Western blotting. Blots were first probed with anti-phosphorylated antibodies and then reprobed with anti-Erk or anti-pan-Akt antibodies, anti-a-tubulin antibody. The figure shows data from one independent experiment (n = 3; the original blots are shown in the Supplementary Information file). (B) The ratio of signal derived from phosphorylated Erk over Erk bands was calculated (n = 3; *p < 0.05 in relation to corresponding “0% FBS”; Wilcoxon t-test). (C) The ratio of signal derived from phosphorylated Akt over Akt bands was calculated (n = 3; *p < 0.05 in relation to corresponding “0% FBS”; Wilcoxon t-test). (D) SH-SY5Y cells in serum-free culture medium (“0% FBS”) with or without BDNF (~ 1 nmol) or GSB-106 (100 nmol) were also pre incubated in the presence or absence of K252a (500 nmol, 60 min). Blots were first probed with anti-phosphorylated antibodies and then reprobed with anti-Erk or anti-pan-Akt antibodies, anti-a-tubulin antibody. The figure shows data from one independent experiment (n = 3; the original blots are shown in the Supplementary Information file). (E) SH-SY5Y cells in serum-free culture medium (“0% FBS”) with or without BDNF (~ 1 nmol) or GSB-106 (100 nmol) were also pre incubated with or without PD98059 (50 µmol, 60 min). Blots were first probed with anti-phosphorylated Erk (pErk) antibody and then reprobed with anti-Erk antibody, anti-a-tubulin antibody. The figure shows data from one independent experiment (n = 3; the original blots are shown in the Supplementary Information file). (F) SH-SY5Y cells in serum-free culture medium (“0% FBS”) with or without BDNF (~ 1 nmol) or GSB-106 (100 nmol) were also pre incubated with or without LY290042 (50 µmol, 60 min). Blots were first probed with anti-phosphorylated pan-Akt (pAkt1/2/3) antibody and then reprobed with anti-pan-Akt antibody, anti-a-tubulin antibody. The figure shows data from one independent experiment (n = 3; separation bars indicate non-contiguous lanes on the same image acquisition (the original blots are shown in the Supplementary Information file).