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. Author manuscript; available in PMC: 2012 Jun 18.
Published in final edited form as: J Neural Transm (Vienna). 2011 May 7;118(5):795–808. doi: 10.1007/s00702-011-0653-2

Figure 3. Detection of full-length Lrrk2 protein in isolated monocytes, B-cells and T-cells.

Figure 3

(A-F) Peripheral blood mononuclear cells (PBMC; A-B) were obtained by Ficoll separation from healthy adult controls (n=5) and separated from erythrocytes (RBC) and granulocytes. PBMC were further processed using antibodies to CD14 (monocyte marker; in C), CD4 and CD8 (both T-cell markers; in panels D-E), and to CD19 (B-cell marker; E), which were conjugated to and immobilized by magnetic beads (see Materials and Methods) (Vranjkovic et al. 2011). Isolated subpopulations of leukocytes were lysed and then subjected to denaturing SDS/PAGE under reducing conditions, followed by Western blotting. Transgenic flies expressing full-length hLRRK2 cDNA (tg) and their wild-type, non-transgenic littermates (wt) were used as positive and negative controls, respectively. EBV-transformed, B-cell derived lymphoblasts from healthy control and Parkinson disease donors (n=5) were run in parallel as additional controls (panel F). Immunoblot in A was probed with affinity-purified, polyclonal anti-human Lrrk2 antibody (HL-2) raised against residues 2508-2527; probing with a monoclonal rabbit anti-Lrrk2 antibody [MJFF-1] is shown in B. Note, polyclonal anti-Lrrk2 antibody NB300-268 was used for membranes C to F. Anti-acid β-glucosidase was used as an independent marker of peripheral monocytes (see bottom panel in C). All membranes were stripped and reprobed with anti-β-actin antibody as loading control. Note, the robust and specific detection of endogenous, full-length Lrrk2 proteins (245-260 kDa) and higher molecular weight (HMW) species thereof in human PBMC, monocytes, B-cells and T-cells. There, CD19+ B-cells showed a slightly stronger signal for Lrrk2 when compared to isolated CD4+ T-cells enriched from the same donor (E).