Anti-RAB8A (phospho T72) antibody [MJF-R20]

• WB

Unknown

Western blot - Anti-RAB8A (phospho T72) antibody [MJF-R20] (AB230260)

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

The LRRK2 pathogenic mutation R1441C increases LRRK2 activity and markedly elevates Rab8 phosphorylation in MEF (mouse embryonic fibroblasts).

The expression profile is consistent with the literature (PMID : 29127256).

The cell lysates were kindly provided by our collaborator, Dr. Dario Alessi.

All lanes:

Western blot - Anti-RAB8A (phospho T72) antibody [MJF-R20] (ab230260) at 1/1000 dilution

Lane 1:

Wild-type MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate at 20 µg

Lane 2:

Wild-type MEF (mouse embryonic fibroblast (immortalized)) treated with 100 nM MLi-2 for 90 minutes, whole cell lysate at 20 µg

Lane 3:

LRRK2 [R1441C] knock-in MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate at 20 µg

Lane 4:

LRRK2 [R1441C] knock-in MEF (mouse embryonic fibroblast (immortalized)) treated with 100 nM MLi-2 for 90 minutes, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 24 kDa

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Exposure time: 1min

• WB

PubMed

Western blot - Anti-RAB8A (phospho T72) antibody [MJF-R20] (AB230260)

Blocking buffer : 5% NFDM/TBST.

Dilution buffer : 5% BSA/TBST.

The LRRK2 pathogenic mutation Y1699C increases LRRK2 activity and markedly elevates Rab8 phosphorylation in MEF (mouse embryonic fibroblasts).

The results show that this antibody recognizes other LRRK2-phosphorylated Rab proteins (Rab3A, Rab10, and Rab35). The phospho-threonine site in Rab8 (T72) differs from that of Rab3A (T86); Rab10 (T73); and Rab43 (T82).

The images were kindly provided by our collaborator, Dr. Dario Alessi, and have been published (PMID : 29127256).

All lanes:

Western blot - Anti-RAB8A (phospho T72) antibody [MJF-R20] (ab230260) at 1/1000 dilution

Lane 1:

HEK-293 cells transfected with LRRK2 [Y1699C] and HA-tagged Rab3A expression vectors, were treated with150 nM MLi-2 for 90 minutes, whole cell lysate, 100 ng

Lane 2:

HEK-293 cells transfected with LRRK2 [Y1699C] and HA-tagged Rab8A expression vectors, were treated with150 nM MLi-2 for 90 minutes, whole cell lysate, 100 ng

Lane 3:

HEK-293 cells transfected with LRRK2 [Y1699C] and HA-tagged Rab10 expression vectors, were treated with150 nM MLi-2 for 90 minutes, whole cell lysate, 100 ng

Lane 4:

HEK-293 cells transfected with LRRK2 [Y1699C] and HA-tagged Rab35 expression vectors, were treated with150 nM MLi-2 for 90 minutes, whole cell lysate, 100 ng

Lane 5:

HEK-293 cells transfected with LRRK2 [Y1699C] and HA-tagged Rab43 expression vectors, were treated with150 nM MLi-2 for 90 minutes, whole cell lysate, 100 ng

Secondary

All lanes:

IRDye 800CW secondary antibody at 1/25000 dilution

Predicted band size: 24 kDa

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• Dot

Unknown

Dot Blot - Anti-RAB8A (phospho T72) antibody [MJF-R20] (AB230260)

Dot Blot - Anti-Rab8A (phospho T72) antibody [MJF-R20] (ab230260) used at a 1/1000 dilution.

Lane 1 : Rab8 (phospho T72) peptide.

Lane 2 : Rab8 non-phospho peptide.

ab97051 secondary antibody used at a 1/100,000 dilution.

Exposure time : 32 seconds.

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

• WB

CiteAb

Western blot - Anti-RAB8A (phospho T72) antibody [MJF-R20] (AB230260)

RAB8A (phospho T72) western blot using anti-RAB8A (phospho T72) antibody [MJF-R20] ab230260. Publication image and figure legend from Chen, C., Soto, G., et al., 2020, Elife, PubMed 33006315.

ab230260 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab230260 please see the product overview.

LRRK2 RC mutation increases synaptic glutamate receptor content in the striatum.(A) Schematic diagram of LRRK2 protein highlighting the armadillo repeats (ARM), ankyrin (ANK) repeats, Ras of complex (ROC), C-terminal of ROC (COR), kin (KIN), and WD40 domains. Knock- in mice expressing the R1441C and G2019S mutations found in the ROC and kinase domains respectively, crossed with either Drd1-Tomato or Drd2-eGFP mouse lines. (B) Workflow schematic for subcellular fractionation of striatal homogenate for the enrichment of postsynaptic density fraction (PSD). (C) Representative western blot analysis of the subcellular fractionation results, showing supernatant (S1), crude synaptosomal preparation (P2), PSD, and Triton soluble fractions (TSF). (D) Western blot analysis of +/+, +/RC, and +/GS P2 striatal fractions probed for p-PKA substrates, pS845 GluA1, total GluA1, pT72Rab8A, total Rab8A, pT73Rab10, total Rab10, and PSD95. (E) Western blot analysis of +/+, +/RC, and +/GS mice probed for GluA1, p-PKA, and PSD95. S1 and PSD fractions are shown. (F-G) Quantification of GluA1 and p-PKA proteins in PSD fractions normalized to PSD95. Summary graphs reflect the mean, error bars reflect SEM. *p<0.05, Tukey post-hoc test following one-way ANOVA.Figure 1—source data 1.Numerical data of the graphs in Figure 1.Numerical data of the graphs in Figure 1.LRRk2 mutations do not alter PSD95 levels.(A–B) Supernatant (S1), crude synaptosomal preparation (P2), PSD, and Triton soluble fractions (TSF) of wild type, +/RC and +/GS striata were run on the same blot. Ponceau staining shows similar loading across genotypes at a given fraction. Western blot analysis of the fractions described in A, probed for PSD95 and Homer-1. (C) Quantification of PSD95 band intensities normalized to Homer-1. Summary graphs represent the mean, while error bars SEM. (D) Quantification of PSD95 band intensities expressed as a fraction of the intensity of PSD95 wild type in each one of the S1, P2, and PSD fractions.Figure 1—figure supplement 1—source data 1.Numerical data of the graphs in Figure 1—figure supplement 1.Numerical data of the graphs in Figure 1—figure supplement 1.

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