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1.
Figure 4

Figure 4. S935A mutation does not affect LRRK2 dimerization.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

(A, B) FLAG-LRRK2 WT or S935A mutant construct was co-transfected with HA-LRRK2 constructs. The Co-IP assay was performed using either anti-HA (A) or anti-FLAG (B) antibody, followed by Western blot analysis using the indicated antibodies.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.
2.
Figure 7

Figure 7. PKA Phosphorylation, 14-3-3 binding of LRRK2, and the effect of common familial mutations of LRRK2 in 14-3-3 binding.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

A schematic model showing PKA (or other kinase) phosphorylation of S910/S935, dimeric14-3-3 binding of LRRK2 at pS910/pS935 sites in wild type LRRK2; PD-linked mutations R1441G, Y1699C or G2019S abolishes or reduces phosphorylation of S910/S935 and impairs 14-3-3 binding. In addition, we propose that dimeric 14-3-3 bind to pS910 and pS935 in the same LRRK2 molecule and binding of 14-3-3 plays little role in LRRK2 dimer formation.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.
3.
Figure 2

Figure 2. Identification of 14-3-3s as LRRK2 interaction proteins.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

(A) Coomassie blue staining showing the affinity-purified FLAG-LRRK2 and its binding proteins from FLAG-LRRK2 transgenic brain. The positions of the FLAG-LRRK2 and 14-3-3 bands are labeled with arrows. Wild-type mouse brain was used as the control for background proteins during purification. (B) Western blot analysis was performed to detect the presence of various 14-3-3 isoforms in association with the affinity purified FLAG-LRRK2 protein. Antibodies against specific isoforms of 14-3-3 were used. Wild type mouse tissue was used as the control. (C) FLAG-LRRK2 and different Myc-14-3-3 isoform plasmids were co-transfected into HEK-293T cells and Immunoprecipitation was performed using anti-Myc antibody, followed by the analysis of protein levels using indicated antibodies. (D) FLAG-LRRK2 plasmid was transfected into HEK-293T cells and transfected cell lysate was incubated with different purified GST-14-3-3 isoforms as indicated. The GST-pull down assay was assayed by Western blot analysis of the FLAG-LRRK2.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.
4.
Figure 3

Figure 3. 14-3-3 binding to LRRK2 S935 is phosphorylation-dependent.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

(A, B) FLAG-LRRK2 Wt or S935A mutant plasmid was transfected with or without Myc-14-3-3γ into HEK-293T cells. Co-immunoprecipitation experiments were performed using either anti-myc (A) or anti-FLAG (B) antibody, followed by Western blot analysis using the indicated antibodies. (C) Sepharose bead-conjugated GST-14-3-3γ was incubated with FLAG-LRRK2 transgenic brain lysate in the presence of various concentrations of phosphorylated peptide (pS935) or non-phosphorylated peptide. The binding FLAG-LRRK2 protein was pulled down and analyzed by Western blot analysis using anti-FLAG antibody. (D, E) FLAG-LRRK2 was transfected alone, with Myc-14-3-3γ Wt, with Myc-14-3-3 g K50E mutant, or with Myc-14-3-3γ V181D mutant into HEK-293T cells. The co-IP assay was performed using either anti-Myc (D) or anti-FLAG (E) antibody, followed by Western blot analysis using indicated antibodies. (F) FLAG-LRRK2 was transfected alone, with Myc-14-3-3γ wt or Myc-14-3-3γ dominant negative (DN) mutant (K50E/V181D) into HEK-293T cells. The total FLAG-LRRK2 was immunoprecipitated and analyzed by Western blot using anti-pS935 and anti-FLAG antibodies. The Western blot shows the results from three independent transfections. The quantification of the signals was done by using LI-COR imaging and Odyssey software. The data was analyzed by One-way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.
5.
Figure 5

Figure 5. PD-linked mutants affect S935 phosphorylation and 14-3-3 binding of LRRK2.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

(A) FLAG-LRRK2 WT, S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected into HEK-293T cells. The LRRK2 variants were immunoprecipitated and analyzed by Western blot using anti-pS935 and anti-FLAG antibodies. Quantification of the signals was performed using LI-COR and Odyssey software and the data was analyzed by One-way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments. (B) FLAG-LRRK2-WT, G2019S, R1441G and K1906M was immunoprecipitated from the brain lysates of the corresponding BAC-transgenic mice. The quantification of the signals was performed as described above. The ratio of pS935 signal over total LRRK2 signal is shown. The data was analyzed with One-way ANOVA (* P<0.05, ** P<0.01). Data are presented as mean value (± SEM) from three mice. (C) Myc-14-3-3γ (or control Myc vector) was co-transfected with FLAG-LRRK2 Wt, S935A, R1441G or G2019S mutant plasmid into HEK-293T cells. Co-IP was performed by using anti-Myc antibody. The pulled down LRRK2 variant levels were analyzed by Western blot analysis with the indicated antibodies and quantified using LI-COR and Odyssey system. The data was analyzed by One-way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments. (D) FLAG-LRRK2-WT, S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected individually into HEK-293T cells. Sepharose 4B beads-conjugated GST-14-3-3γ protein was incubated with different transfected cell lysates containing LRRK2-Wt or various mutants. Western blot analysis was performed to determine the LRRK2 variant amount after the GST pull-down assay. The results were quantified as described above. The ratio of pulled down LRRK2 and input LRRK2 was measured and analyzed by One-Way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.
6.
Figure 1

Figure 1. Identification of LRRK2 phosphorylation sites in BAC transgenic brain.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

(A) Table of the identified phosphorylation sites within tryptic peptides from purified brain FLAG-LRRK2 protein. The estimate of the stoichiometry of phosphorylation is based on the ratio of number of MS/MS spectra observed. The exact location of each proteolytic peptide is shown in the position column. (B) Purified brain FLAG-LRRK2 protein was treated with or without Calf-intestinal alkaline phosphatase (CIAP), followed by Western blot analysis with anti-pS935 antibody or anti-FLAG antibody. (C) FLAG-LRRK2 WT or FLAG-LRRK2 S935A mutant plasmid was transfected into HEK-293T cells; FLAG-tagged proteins were then immunoprecipitated and analyzed by anti-pS935 antibody or anti-FLAG antibody via Western blot analysis. (D) FLAG-LRRK2 was purified from different BAC-transgenic tissues and western blot was performed to detect pS935 and total FLAG-LRRK2 levels. No significant difference in the ratio of pS935/total LRRK2 was observed between different tissues. Data are presented as mean value (± SEM) from three mice. (E) FLAG-LRRK2 was purified from 3 months and 18 months transgenic mouse brain and western blot was performed to detect pS935 and total FLAG-LRRK2 levels. No significant difference in the ratio of pS935/total LRRK2 was observed between 3 months and 18 months. The Western blot signals were quantified by using LI-COR and Odyssey software. Data are presented as mean value (± SEM) from three mice.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.
7.
Figure 6

Figure 6. PKA, not LRRK2 itself, can phosphorylate S935 in vitro and within cells.. From: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease.

(A) 0.4 µg of purified GST-LRRK2 fragments (800–1000aa) was incubated with different amount of the PKA catalytic subunit (lane 1: without PKA; lane 2: 2500 units PKA; lane 3: 10,000 units PKA). The reaction was stopped by adding 3× SDS-PAGE sample buffer and Western blot was performed by using anti-pS935 and anti-LRRK2 antibodies. (B) Different concentrations of H-89 (0, 2.5, 5 and 10 µM) were added to the PKA and GST-LRRK2 fragment reaction mixture, and Western blot analyses was performed by using anti-pS935 and anti-LRRK2 antibodies. (C) FLAG-LRRK2 was co-transfected with different amounts of PKA plasmid into HEK-293T cells (The ratio of FLAG-LRRK2 verse PKA plasmid DNA are: 0 in lane 1, 1∶1 in lane 2 and 1∶10 in lane 3). The transfected cell lysate was harvested and analyzed by Western blot using anti-pS935, anti-FLAG, anti-PKA, anti-pGSK and anti-β-actin antibodies. (D) FLAG-LRRK2 was transfected into HEK-293T cells and after 40 hours transfection, 10 µM FSK was added to the cell culture medium and incubated for 30 min. Then the cells were harvested and western blot analysis was performed to study various protein levels by using anti-pS935, anti-FLAG, anti-pGSK and anti-β-actin antibodies. The pS935 LRRK2 and total LRRK2 signals were quantified by using the LI-COR Odyssey software system and the ratio of pS935 and total LRRK2 was calculated and analyzed by One-way ANOVA (** P<0.01). Western blot results of three independent experiments are shown, and data are presented as mean value (± SEM) from three independent experiments. (E) GST-LRRK2 fragment (800–1000aa) protein was incubated with purified FLAG-LRRK2 (from mouse brains) or with PKA catalytic enzyme subunit (as positive control) (lane 1: 2500 units PKA; lane 2: 12.5 nM FLAG-LRRK2; lane 3: 25 nM FLAG-LRRK2; lane 4: 125 nM FLAG-LRRK2; lane 5: 250 nM FLAG-LRRK2) in Kinase assay buffer and western blot was performed by using anti-pS935 and anti-LRRK2 antibodies. The arrows indicated the phosphorylated form of full length LRRK2 and GST-LRRK2 fragment; the asterisks indicated the total full length LRRK2 and total GST-LRRK2 fragment, respectively.

Xianting Li, et al. PLoS One. 2011;6(3):e17153.

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