SH-SY5Y cells were infected with Pin1 siRNA or control siRNA lentiviruses, followed by selection for stable cells with puromycin. Stable Pin1 KD or control SY5Y cells were transfected overnight with a WT tau expression construct in the presence or absence of a Pin1 expression construct (+Pin1 addback) (A and B), with a P301L tau construct in the presence or absence of a Pin1 construct (C and D), with a tau-T231A construct in the presence or absence of a Pin1 construct (E and F), or with a P301L tau-T231A construct in the presence or absence of a Pin1 construct (G and H). Cycloheximide was added to stop new protein synthesis and chased for indicated times in the absence or presence of the proteosome inhibitor MG132. Cells were harvested and cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting with Tau5, anti-Pin1, or anti-actin antibodies (A, C, E, and G). Tau levels were semiquantified from 3 different experiments using ImageQuant and normalized using actin as an internal control (B, D, F, and H). For comparison, tau levels at 0 (before the cycloheximide addition) were defined as 100%. The opposite effects of Pin1 KD on protein stability of WT tau and P301L tau were also obtained in HT1080 cells, as shown by dashed lines in B and D. Error bars represent SD.