Immunofluorescent staining of SH-SY5Y cells fixed and permeablized with 4% PFA and 0.1% Triton X 100 using purified ab133518 at a dilution of 1/200. An Alexa Fluor^® 488 goat anti-rabbit was used as the secondary (ab150077, 1/400) and the sample was stained with DAPI. The negative control is shown in bottom right hand panel - for the negative control, purified ab133518 was used at a dilution of 1/200 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500.

Immunohistochemical staining of paraffin-embedded human kidney with purified ab133518 at a dilution of 1/100. A prediluted HRP polymer for rabbit IgG was used as the secondary and the sample was stained with hematoxylin. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ab133475, at 1/200 dilution, staining LRRK2 in paraffin embedded Human hippocampus tissue using immunohistochemical analysis.

Immunocytochemistry/Immunofluorescence analysis of A431 (Human epidermoid carcinoma cell line) cells labeling LRRK2 (phospho S935) with purified ab133450 at 1/100.

Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: Secondary antibody, ab150113, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

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.

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 - Anti-LRRK2 antibody [MJFF2 (c41-2)] (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).

All lanes : Anti-LRRK2 (phospho S1292) antibody [MJFR-19-7-8] (ab203181) at 1 µg/ml

Lane 1 : GFP-LRRK2 wt transfected HEK293 lysate subjected to immunoprecipitation with GFP trap agarose

Lane 2 : GFP-LRRK2 mutant D2017A transfected HEK293 lysate subjected to immunoprecipitation with GFP trap agarose

Lane 3 : GFP-LRRK2 mutant S1292A/G2019S transfected HEK293 lysate subjected to immunoprecipitation with GFP trap agarose

Lane 4 : GFP-LRRK2 mutant G2019S transfected HEK293 lysate subjected to immunoprecipitation with GFP trap agarose

Lysates/proteins at 10 µg per lane.

Secondary

All lanes : Goat anti-rabbit (IRDye 800) at 1/10000 dilution

Predicted band size: 286 kDa

Observed band size: 313 kDa

Blocking/Dilution buffer: 5% BSA/TBST.

The image is provided by Dr. Jeremy Nicols, Parkinson’s Institute, Sunnyvale, CA, USA.

G2019S mutation results in an increased LRRK2 auto-phosphorylation including S1292 (lane 4).

Observed band size: 286 + 27 (GFP) = 313 kDa.