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. 2021 Jun 21;41(6):BSR20203947. doi: 10.1042/BSR20203947

Figure 3. Phosphorylation state of various signalling pathways upon treatment with high D-glucose and TGF-β1 in PTECs.

Figure 3

(A–C) Image of western blots immunostained for cellular phospho-ERK protein in PTECs under various treatments and time points. (D–F) Data points for cellular phospho-ERK (∼40 kDa) abundance normalised for total protein as mentioned above in Figure 1. TGF-β1 induced ERK phosphorylation significantly, +/- high D-glucose for 60 min, while exclusive high D-glucose treatment phosphorylated ERK at 30 min; *P<0.05. (G–I) Image of Western blots immunostained for cellular phospho-Smad3 protein in PTECs under various treatments and time points; (G and H) Smad3 phosphorylated at serine 423/425; (I and J) Smad3 phosphorylated at serine 204. (J–L) Data points for cellular phospho-Smad3 (∼50 kDa) abundance normalised for total protein. TGF-β1 significantly phosphorylated Smad3, +/- high D-glucose from 15 min onwards; **P<0.01. (M–O) Image of Western blots immunostained for cellular phospho-serine 204 of Smad3 LR protein in PTECs under various treatments and time points. (P–R) Data points for cellular phospho-serine 204 of Smad3 LR protein (∼25 kDa) abundance normalised for total protein. D-Glu+TGF-β1 treatment at 30 min significantly phosphorylated serine 204 compared to L-Glu+TGF-β1 at 30 min; *P<0.05, n=3–4. Black squares ▓ = L-Glu+TGF-β1, black circles ● = D-Glu+TGF-β1, white circles ○ = D-Glu, white squares □ = L-Glu. Error bars indicate SD. Quantification of all phosphorylated proteins in these figures were a result of the light generated by the detection antibody indexed to total protein loaded in each sample. Control = 7 mM D-glucose, Control + TGF-β1 = 7 mM D-glucose + 0.75 ng/ml TGF-β1, D-Glu = 7 mM D-glucose + 18 mM D-glucose, L-Glu = 7 mM D-glucose + 18 mM L-glucose, D-Glu + TGF-β1 = 7 mM D-glucose + 18 mM D-glucose + 0.75 ng/ml TGF-β1, L-Glu + TGF-β1 = 7 mM D-glucose + 18 mM L-glucose + 0.75 ng/ml TGF-β1. This legend key is applicable to all subsequent figures.