Figure 4. Heterologous co-immunoprecipitation confirms an enrichment of the LRRK2 dimer at the membrane.

(A) To optimize the heterologous co-immunoprecipitation (co-IP) system, the amount of myc-LRRK2 DNA was titrated to match the expression levels of GFP-LRRK2. Transfection with 0.5 μg of myc-LRRK2 led to similar levels of LRRK2 protein as 5 μg of GFP-LRRK2.

(B) A schematic depicting possible LRRK2 dimers after transfection with GFP-LRRK2 and myc-LRRK2 plasmids. Co-IP of heterologously tagged constructs allows quantification of GFP-LRRK2/myc-LRRK2 heterodimer, which at equal levels of both proteins will likely represent 50% of total dimer.

(C) Cytosol and membrane fractions from cells co-expressing GFP-LRRK2 and myc-LRRK2 (lanes 1–2) or GFP-LRRK2 and an empty vector (lanes 3–4) were used for IP using a high affinity myc resin. GFP-LRRK2 is co-IPed only in the presence of myc-LRRK2 (lanes 5–9), confirming specificity of IP. Higher levels of GFP-LRRK2 are pulled down from membrane extracts (lane 7), despite lower levels of myc-LRRK2 in the same IP, suggesting an enrichment of LRRK2 dimer at the membrane. Identical results from two independent samples (C₁, C₂) using the same total cytosolic fraction (C_T) illustrate the low variability of this assay.

(D), Levels of LRRK2 heterodimers are 5.7-fold higher in membrane extracts (Mean ± SEM, p = 0.01, n = 3, unpaired t-test). The relative dimer levels were quantified by measuring the band intensity of co-IPed GFP-LRRK2 divided by the intensity of IPed myc-LRRK2. The value for cytosol was arbitrarily set as 1. ** p ≤ 0.01.