Background
Results
LRRK2 phosphorylates p53 in vitro
LRRK2 interacts with and phosphorylates p53 in physiological condition
LRRK2-induced p53 phosphorylation accumulates p53 in the nucleus and activates p21WAF1/CIP1 expression
Discussion
p53 as a LRRK2 kinase substrate
Physiological consequences of p53 phosphorylation by LRRK2
Conclusions
Methods
Plasmids and recombinant proteins
Cell culture and transfection
In vitro kinase assay
Reprogramming of human fibroblasts to induced pluripotent stem cells and their differentiation to dopaminergic neurons
Immunoprecipitation, nuclear fractionation and Western blot
Luciferase assay and reverse transcription –polymerase chain reaction (RT-PCR)
Primer name | Gene | Direction | Sequence | TM (°C) | Product size(bp) |
---|---|---|---|---|---|
hP21-F | P21 | Forward | GTTCCTTGGGAGCCGGAGC | 60 | 417 |
hP21-R | Reverse | GGTACAAGACAGTGACAGGTC | |||
LRRK2-7232 | LRRK2 | Forward | GTCATGATGACAGCACAGC | 65 | 201 |
LRRK2-7440 | Reverse | CTCTTACTCAACAGATGTTCGTCTC | |||
pMycXhoI-R | Myc tag | Reverse | CTTCTGAGATGAGTTTTTGTTCCT CGAC | 57.3 | 254a
|
b-actin-F | β-actin | Forward | CGCCGCCAGCTCACCATG | 62.5 | 120 |
b-actin-R | Reverse | CACGATGGAGGGGAAGACGG |