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. Author manuscript; available in PMC: 2021 Feb 1.

Published in final edited form as: FASEB J. 2020 Jan 9;34(2):3179–3196. doi: 10.1096/fj.201901777R

Figure 3. — A. Phospho-tau T231 can be used as a readout of PPP5C cellular activity. (i) Phospho-tau T231 was assessed via immunoblot of HEK293 cells which were engineered to modestly overexpress PPP5C (~2- fold) (23). Compared to wild-type (WT) HEK293 cells, in PPP5C over-expressed cells, phospho-tau T231 was nearly abolished. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. n = 3 independent experiments. B. S100A6 activates PPP5C in endothelial cells. (i) S100A6 over-expressing cells express decreased phospho-tau T231. Immunoblot of doxycycline-inducible S100A6 PMVECs reveal ~ 2-fold increase in S100A6 expression in the presence of doxycycline, and a corresponding decrease in phospho-tau T231. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; S100A6 10 kDa; β actin 42 kDa. n = 3 independent experiments. (iii) The decrease in phospho-tau T231 by S100A6 over-expression is dependent upon PPP5C. Doxycycline-inducible S100A6 PMVECs were treated with PPP5C siRNA or scrambled control and phospo-tau T231 expression measured by immunoblot. In the presence of scrambled control, S100A6 over-expressed cells revealed little phospho-tau T231 which was reversed upon knock-down of PPP5C. (iv) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; PPP5C ~58 kDa; S100A6 10 kDa; β actin 42 kDa. n = 5 independent experiments. C. Suppression of endogenous S100A6 expression results in increased phospho-tau T231 in PMVECs. (i) PMVECs were treated with S100A6 siRNA or scrambled control. In both the absence of I_SOC activation (control) and following I_SOC activation, phospho-tau T231 was increased in cells treated with S100A6 siRNA as compared to scrambled control with no change in total tau levels. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; β actin 42 kDa. n = 3 independent experiments. D. FKBP51 over-expressing cells express decreased phospho-tau T231. (i) Immunoblot of doxycycline-inducible FKBP51 PMVECs reveal ~ 3-fold increase in FKBP51 expression in the presence of doxycycline, and a corresponding decrease in phospho-tau T231. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; FKBP51 ~51 kDa; β actin 42 kDa. n = 3 independent experiments. (iii) The decrease in phospho-tau T231 by FKBP51 over-expression is dependent upon S100A6. Doxycycline-inducible FKBP51 PMVECs were treated with S100A6 siRNA or scrambled control and phospho-tau T231 expression measured by immunoblot. In the presence of scrambled control, FKBP51 over-expressed cells revealed little phospho-tau T231 which was reversed upon S100A6 suppression. (iv) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; S100A6 10 kDa; FKBP51 ~51 kDa; β actin 42 kDa. n = 5 independent experiments.

Figure 3. — A. Phospho-tau T231 can be used as a readout of PPP5C cellular activity. (i) Phospho-tau T231 was assessed via immunoblot of HEK293 cells which were engineered to modestly overexpress PPP5C (~2- fold) (23). Compared to wild-type (WT) HEK293 cells, in PPP5C over-expressed cells, phospho-tau T231 was nearly abolished. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. n = 3 independent experiments. B. S100A6 activates PPP5C in endothelial cells. (i) S100A6 over-expressing cells express decreased phospho-tau T231. Immunoblot of doxycycline-inducible S100A6 PMVECs reveal ~ 2-fold increase in S100A6 expression in the presence of doxycycline, and a corresponding decrease in phospho-tau T231. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; S100A6 10 kDa; β actin 42 kDa. n = 3 independent experiments. (iii) The decrease in phospho-tau T231 by S100A6 over-expression is dependent upon PPP5C. Doxycycline-inducible S100A6 PMVECs were treated with PPP5C siRNA or scrambled control and phospo-tau T231 expression measured by immunoblot. In the presence of scrambled control, S100A6 over-expressed cells revealed little phospho-tau T231 which was reversed upon knock-down of PPP5C. (iv) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; PPP5C ~58 kDa; S100A6 10 kDa; β actin 42 kDa. n = 5 independent experiments. C. Suppression of endogenous S100A6 expression results in increased phospho-tau T231 in PMVECs. (i) PMVECs were treated with S100A6 siRNA or scrambled control. In both the absence of I_SOC activation (control) and following I_SOC activation, phospho-tau T231 was increased in cells treated with S100A6 siRNA as compared to scrambled control with no change in total tau levels. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; β actin 42 kDa. n = 3 independent experiments. D. FKBP51 over-expressing cells express decreased phospho-tau T231. (i) Immunoblot of doxycycline-inducible FKBP51 PMVECs reveal ~ 3-fold increase in FKBP51 expression in the presence of doxycycline, and a corresponding decrease in phospho-tau T231. (ii) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; FKBP51 ~51 kDa; β actin 42 kDa. n = 3 independent experiments. (iii) The decrease in phospho-tau T231 by FKBP51 over-expression is dependent upon S100A6. Doxycycline-inducible FKBP51 PMVECs were treated with S100A6 siRNA or scrambled control and phospho-tau T231 expression measured by immunoblot. In the presence of scrambled control, FKBP51 over-expressed cells revealed little phospho-tau T231 which was reversed upon S100A6 suppression. (iv) Quantitation of phospho-tau T231 was assessed via densitometry of phospho-tau T231 and total tau bands and the normalized phospho-tau T231/total tau ratio determined. Molecular weights observed: phospho-tau T231 ~55 kDa; total tau ~50 kDa; S100A6 10 kDa; FKBP51 ~51 kDa; β actin 42 kDa. n = 5 independent experiments.