The Journal of Neuroscience, January 17, 2007, ():
Activation of PAR-1 Kinase and Stimulation of Tau Phosphorylation by Diverse Signals Require the Tumor Suppressor Protein LKB1
J. Neurosci. Wang et al.
27: 574
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Figure S1. Additional data supporting the specificity of LKB1 on PAR-1 T408 phosphorylation. (A) Control experiment showing no enhancement of PAR-1(T408A) protein level by LKB1 co-expression. (B) Western blot analysis comparing the levels of p-PAR-1 in transgenic animals overexpressing LKB1 or PAR-1. Tubulin serves as a loading control. Asterisks and arrow mark endogenous and exogenous PAR-1, respectively. (C) A bar graph showing quantification of the reduction of p-PAR-1 level by LKB1 RNAi. (D) Western blot analysis showing the effect of dMARKK/TAO1 or mMARKK/TAO1 co-expression on p-PAR-1 level. Tubulin serves as a loading control.
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Figure S2. Detection of potential LKB1/PAR-1 complex formation by immunoprecipitation. HEK293 cells were transfected with the indicated constructs. Cell lysates were immunoprecipitated with anti-Flag agarose beads to isolate LKB1 and the immunocomplexes were probed by Western blotting with the corresponding anti-epitope tag antibodies to detect the presence of PAR-1 or STRAD. Note that STRAD was readily IPed by LKB1, but no PAR-1 protein was detected in the IP complex even after longer exposure. In the Western blot analysis of cell lysates, a non-specific band (marked with an asterisk) right below the His-STRAD band (marked with an arrow) was detected by the anti-His antibody.
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Figure S3. Western blot analysis showing that LKB1 overexpression enhances phosphorylation of endogenous tau at 12E8 sites, whereas LKB1 RNAi has the opposite effect. W RNAi and dPink1 RNAi were used as RNAi controls.
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Figure S4. Additional control experiment supporting the specificity of the LKB1 RNAi effect in blocking APP-induced PAR-1 and tau phosphorylation. (A) Control experiments showing that W RNAi has little effect on APP-induced PAR-1 T408 site phosphorylation. (B) Control experiment showing lack of effect on APP-induced tau phosphorylation at 12E8 sites by W RNAi. After normalizing the amount of actin as loading control, the level of 12E8 phosphorylation in h-tau(wt)/APP and h-tau(wt)/APP/W RNAi samples is comparable, which is approximately 2.5 fold higher than the level in h-tau(wt) sample. (C) Control experiment showing elevation of p38 MAPK phosphorylation by high salt treatment.
