Evaluation of DRD1 intracellular and extracellular level by BPA upon dopamine treatment in SH-SY5Y-DRD1 cells untransduced or transduced by WT or G2019S LRRK2

(C and D) DRD1 localization at basal conditions (C) and upon 15 minutes (D) of dopamine treatment of SH-SY5Y-DRD1 cells transduced or not by alpha-synuclein WT or A53T. After agonist treatment the cells were fixed and incubated with the different primary antibodies (anti-FLAG for DRD1 and ab133474) and with Alexa647-conjugated secondary antibody (red) or Alexa488-conjugated secondary antibody (green) for DRD1 or LRRK2 respectively. Scale bars = 10μm.

 

Lane 1: Wild type A549 whole cell lysate (20 µg)
Lane 2: Wild type MEF whole cell lysate (20 µg)
Lane 3: LRRK2 knockout A549 whole cell lysate (20 µg)
Lane 4: LRRK2 knockout MEF whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab133474 observed at 286 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab133474 was shown to recognize LRRK2 in wild type A549 and MEF cells along with additional cross reative bands. Whilst signal was not seen in LRRK2 knockout cells. Wild-type and LRRK2 knockout samples were subjected to SDS-PAGE. Ab133474 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 10000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Wild-type and LRRK2 knockout MEF and A549 cells were provide as a generous gift from Professor Dario Alessi, MRC Protein Phosphorylation and Ubiquitination Unit (University of Dundee).

ab133474 staining LRRK2 in Neuro-2a (mouse neuroblastoma cell line) cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/500). ab150077 was used as the secondary antibody (1/1000). Tubulin stained using ab7291 (1/1000) and ab150120 (1/1000) as the secondary. Nuclear counter stained with DAPI.

Immunoprecipitation to verify the interaction of LRRK2 and ArfGAP1 in vivo. LRRK2 interacts with ArfGAP1 in brain extracts derived from wild-type mice following immunoprecipitation with ab133474, a LRRK2-specific monoclonal antibody (MJFF-2/c41-2), whereas ArfGAP1 is not immunoprecipitated in extracts derived from LRRK2 knockout mice

Protein extracts were prepared from the cerebral cortex of adult wild-type and LRRK2 knockout mice (with targeted deletion of exon 41 of the LRRK2 gene) by homogenization in TNE buffer (10 mM Tris-HCL pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1× phosphatase inhibitor cocktail 1 and 2, 1× Complete Mini protease inhibitor cocktail). Protein concentration was determined by BCA assay. Brain extracts (10 mg protein) were combined with 50 µl Protein G-Dynabeads pre-incubated with rabbit anti-LRRK2 (5 µg; MJFF2/c41-2; Abcam, Inc.), rabbit anti-ArfGAP1 (3 µg) or rabbit IgG (3 µg) antibodies followed by overnight incubation at 4°C. Dynabead complexes were sequentially washed twice with TNE buffer and twice with TBS buffer (10 mM Tris-HCL pH 7.4, 150 mM NaCl). Immunoprecipitates were eluted by heating at 70°C for 10 min, resolved by SDS-PAGE and subjected to Western blot analysis.

All lanes : Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474) at 1/10000 dilution

Lane 1 : HEK293 cell lysate transfected with 3*Flag vector
Lane 2 : HEK293 cell lysate transfected with 3*Flag full length wild type LRRK2

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 286 kDa