Fig 1: Phosphorylation levels of released hTau at specific pSites are decreased after GSK-3β inhibition.ReNCell cultures (14 DIV) were incubated with SB (10μM) for 2hrs before 50mM KCl stimulation (1 hour). (A-E) Representative western blot images showing phosphorylated hTau from cell lysate and IP-CM at pSites recognized by AT270 (A), AT8 (B), AT100 (C), AT180 (D), and PHF-1 (E). (A’-E’) Quantification of phosphorylated hTau released to CM at pSites detected by AT270 (A’), AT8 (B’), AT100 (C’), AT180 (D’), and PHF-1 (E’). (A”-E”) Quantification of phosphorylated hTau in cell lysate at pSites detected by AT270 (A”), AT8 (B”), AT100 (C”), AT180 (D”), and PHF-1 (E”). Data from 3 independent experiments. Tau bands between 50 and 120kDa were quantified relative to β-actin in cell lysate. Error bars indicate Mean ± SEM. Student t-test, *p<0.05, **p<0.01, ***p<0.001.
Fig 2: Intracellular and released hTau from human ReNCell culture.(A) A representative western blot (WB) image showing intracellular tau in lysate and released tau immunopurified from conditioned media (IP-CM). Tau was released to the culture media after 50mM KCl stimulation for 1 hr at 14 DIV. (B and C) Tau bands quantified in CM and lysate were between 50 and 120kD. Quantification of HT7 intensity in IP-CM from control and KCl treated sample groups (B). (C) shows quantification of intracellular hTau in cell lysate. Data from 3 independent experiments. Student t-test, *p<0.05.
Fig 3: Dephosphorylation of release hTau from ReNCell culture at TAU-1 sites.(A) A representative western blot (WB) image probed with TAU-1 antibody showing intracellular tau in lysate and released tau immunopurified from conditioned media (IP-CM). Tau was released to the culture media after 50mM KCl stimulation for 1 hr at 14 DIV. (B and C) Tau bands quantified in lysate and CM were between 50 and 120kD. Quantification of HT7 intensity in IP-CM from control and KCl treated sample groups (B). (C) shows quantification of intracellular hTau in cell lysate. Data from 3 independent experiments. Student t-test, *p<0.05.
Fig 4: Depolarization-dependent hTau release from differentiated human ReNCell.(A) ELISA analysis showing released hTau in conditioned media (CM). ReNCell cultures were treated with 100uM AMPA and 50mM KCl for 1 hr. Tau release was normalized to total protein in cell lysate. Data from 3 independent experiments. (B) Intracellular hTau levels were not altered by AMPA and KCl stimulation. Error bars indicate mean ± SEM. Student t-test, *p<0.05.
Fig 5: Phosphorylation profiles of intracellular and released hTau from ReNCell.(A-E) Representative western blot images showing phosphorylated hTau from cell lysate and IP-CM at pSites recognized by AT270 (A), AT8 (B), AT100 (C), AT180 (D), and PHF-1 (E). (A’-E’) Quantification of phosphorylated hTau released to CM at pSites detected by AT270 (A’), AT8 (B’), AT100 (C’), AT180 (D’), and PHF-1 (E’). (A”-E”) Quantification of phosphorylated hTau from cell lysate at pSites detected by AT270 (A”), AT8 (B”), AT100 (C”), AT180 (D”), and PHF-1 (E”). Data from 3 independent experiments. Tau bands between 50 and 100kDa were quantified relative to β-actin in cell lysate. Error bars indicate Mean ± SEM. Student t-test, *p<0.05, **p<0.01, ***p<0.001.
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