Anti-LRRK2 (phospho S935) 抗体 [UDD2 10(12)]

• WB

PubMed

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

Inhibitor-induced dephosphorylation of kinase-inactive LRRK2.

HEK-293 cells transfected with wild-type, or mutated LRRK2 (K1906A, K1906M, D1994A, D1994N, D2017A, S2032A or T2035A) were treated with 3 μM LRRK2-IN-1 (LRRK2 inhibitor) for 30 min. The phosphorylation of LRRK2 at Ser910, Ser935, or Ser955 was examined by immunoblotting. This showed that the responses of LRRK2 to the inhibitor varied among mutants.

LRRK2 (phospho S935) was detected using ab133449.

LRRK2 (phospho S935) was detected using ab133450.

LRRK2 (phospho S955) was detected using ab169521.

All lanes:

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (ab133450)

Predicted band size: 286 kDa

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Ito et al PLoS One. 2014 May 16;9(5):e97988. doi: 10.1371/journal.pone.0097988. eCollection 2014. Fig 3. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

Unknown

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

All lanes:

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (ab133450) at 1/1000 dilution

Lane 1:

GFP LRRK2 lysate at 5 µg

Lane 2:

GFP LRRK2 S910A lysate at 5 µg

Lane 3:

GFP LRRK2 S935A lysate at 5 µg

Lane 4:

LRRK2 WT MEF lysate at 20 µg

Lane 5:

LRRK2 WT MEF lysate from LRRK2 IN1 treated cells at 20 µg

Lane 6:

LRRK2 KO MEF lysate at 20 µg

Lane 7:

LRRK2 KO MEF lysate from LRRK2 IN1 treated cells at 20 µg

Lane 8:

Lymphoblastoid lysate at 30 µg

Lane 9:

Lymphoblastoid lysate from LRRK2 IN1 treated cells at 30 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 286 kDa

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• WB

Lab

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (ab133450) at 1/5000 dilution

Lane 1:

WT-LRRK2 cell lysate - untreated at 10 µg

Lane 2:

WT-LRRK2 cell lysate - treated with Lambda phosphatase at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 286 kDa,39 kDa

Observed band size: 286 kDa,39 kDa

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• WB

Lab

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (ab133450) at 1/1000 dilution

All lanes:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 286 kDa

Observed band size: 286 kDa

false

• WB

CiteAb

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

Western Blotting using Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)], ab133450. Publication image from Cookson, M. R. et al., 2014, Nat Commun, 25500533. Legend direct from paper.

Casein kinase 1 alpha (CK1α) is a kinase regulator of LRRK2a) Schematic of the domain organization and location of LRRK2 constitutive phosphorylation sites. Domains include ankyrin (Ank), leucine-rich-repeat (LRR), Ras of complex proteins (ROC), C-terminal of ROC (COR), kinase and WD40 domains. Constitutive phosphorylation sites are clustered upstream of the LRR domain and crucial for binding to 14-3-3 proteins. Pathogenic mutations, shown in red (R1441C, Y1699C and G2019S; N1437S not shown), S910A/S935A, T1348N and K1906M, shown in black, are designed mutants used to block 14-3-3 binding, GTP/GDP binding and kinase activity respectively.b) RNAi screen against kinases to identify kinase regulators of LRRK2 at S935. The screen was performed in duplicate per siRNA pool and each value of ratio pS935/LRRK2 was converted with a Z-transformation, adjusted for date of assay. Hits were identified if both replicates were 3 standard deviation Z away from mean. CSNK1A1 and WEE1 were two candidates with adjusted Z < −3.0 in both duplicates (bottom left grey box).c) Western blot example from the RNAi screen identifying CSNK1A1 as the candidate kinase for S935 LRRK2. Recombinant LRRK2, purified from cells pre-treated with DMSO or LRRK2-IN1, were included in each blot as loading control to allow for normalization across blots.d) CSNK1A1 validated using single siRNAs and pooled siRNAs. Three of four single CSNK1A1 siRNAs showed that when CK1α was knocked down, S935 phosphorylation was also reduced. Representative blots from 3 independent experiments. NTC – non-targeting control, single CSNK1A1 siRNAs - #1, 2, 3, 4 (used at 6.25nM final concentration).e) Quantitation of blots in 1d. Graph shows mean +/− SEM (n=3) for relative CK1α and phosphorylated LRRK2 signals.

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• WB

CiteAb

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

Western Blotting using Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)], ab133450. Publication image from Cookson, M. R. et al., 2014, Nat Commun, 25500533. Legend direct from paper.

CK1α directly phosphorylates LRRK2 in vitro and in vivoa) LRRK2 is a substrate of CK1α. CK1α phosphorylates LRRK2, in vitro, at S910, S935, S955 and S973 sites. Representative blots of 3 independent experiments.b) Consensus sequence of CK1 phosphorylation. The S/Tp-X-X-S/T is the canonical phosphorylation motif42. Alternative phosphorylation motif of CK1 consist of an SLS motif followed by an acidic cluster in positions n+7 (underlined43). Sequence analysis of LRRK2 shows that serines 910, 935 and other constitutively phosphorylated serines at 973/975/976 is a weak consensus site for canonical and non-canonical CK1α phosphorylation. Phosphorylated serines of LRRK2 are shown in red.c) IC261 but not LRRK2-IN1, inhibited CK1α phosphorylation of LRRK2 in vitro. Concentrations of inhibitors used were 50 mM IC261 and 100 nM LRRK2-IN1. Results were consistent even when higher concentration of inhibitor, 100 mM IC261 and 1 µM LRRK2-IN1, was used (Supplementary Fig. 4b). Representative blots of 3 independent experiments.d) Quantitation of blots in 3c. Graph shows mean +/− SEM. Statistical significance tested with two-way ANOVA with Bonferroni post-hoc test (* p<0.05; ** p<0.01; n.s. = not significant).e) LRRK2 is dephosphorylated at S910, S935, S955 and S973 upon knockdown with CSNK1A1 siRNA in a LRRK2-kinase independent manner, as both WT and K1906M is dephosphorylated to the same extent.f) CSNK1A1 siRNA knockdown samples described in 3e were subjected to LC-MS/MS analysis for phospho-peptide mapping. The XIC peak area extracted from the LC-MS/MS data was used to calculate the relative abundance of the detected phospho-peptide in different conditions. Graph shows the quantitative loss of ~70–80%, of pS908, pS910, pS935, pS955, pS973 and pS976 from CSNK1A1 compared to NTC siRNA samples for both WT (filled circles) and K1906M (open circles).

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• WB

CiteAb

Western blot - Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (AB133450)

Western Blotting using Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)], ab133450. Publication image from Cookson, M. R. et al., 2014, Nat Commun, 25500533. Legend direct from paper.

Pharmacological inhibition of CK1α results in loss of pS935 and 14-3-3 bindinga) Inhibition of with CK1, but not CK2, specific kinase inhibitors causes dephosphorylation of pS935 of wildtype (WT) and K1906M in a dose dependent manner. Representative blots of 3 independent experiments.b) Dose response curve of IC261 on phosphorylation of S935 quantified from 2a. IC50 for WT = ~176 µM, IC50 for K1906M = 152 µM. Graph shows mean +/− SEM (n=3).c) CK1α inhibition with 200 µM IC261 abolishes LRRK2/14-3-3 interaction. Representative blots from 4 independent experiments.d) Quantitation of blots in 2c. Graph shows mean +/− SEM (n=3). Statistical significance tested with two-way ANOVA with Bonferroni post-hoc test (** p<0.01; ****p<0.0001).e) CK1α inhibition with IC261 in 14 DIV neurons showed a dose-dependent decrease of endogenous pS935. Representative blots from 3 independent experiments.f) Quantitation of blots in 2e. Graph shows mean +/− SEM.g) Experimental overview of acute brain ex vivo experiment. Coronal brain slices of 1mm thickness were prepared, and slices from one half of the brain from the same animal were treated with DMSO and the other half with either IC261 (#1) or LRRK-IN1 (#2).h) Adult non-transgenic wildtype mouse (4–9 weeks) acute brain ex vivo slices treated with DMSO, 1 µM LRRK2-IN1 and 300 µM IC261 for 2 hrs. S935 phosphorylation was reduced upon treatment with LRRK2-IN1 and IC261.i) Quantitation of blots in 2h. Graph shows mean +/− SEM (n=7). Statistical significance tested with paired t-test comparing treatments within a group (* p<0.05; **p<0.01).

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