Mouse v5

Drosophila S2 cells were cultured in M3 media (Sigma) with 10% Insect medium supplement (Sigma). Transfection was carried out with Effectene reagent (Qiagen) according to manufacturer’s instructions. Total of 1–2 μg DNA was used for each transfection. For immunoprecipitation, cells were lysed in 0.1% CHAPS buffer, and the lysates were precleared by incubating with protein G-sepharose beads (Roche) for 1 h at 4 °C. The G-sepharose beads were immunoprecipitated with anti-Myc (Abcam) or anti-Flag (Sigma) at 4 °C for 1 h. The immunoprecipitates captured by protein G-sepharose were incubated with the clear lysates overnight at 4 °C. Immunoprecipitates were washed three times in cold IP buffer. The samples were boiled in protein loading buffer at 94 °C for 5 min and then subjected to SDS-PAGE. Western blot was performed with mouse Flag (Sigma) or mouse V5 at 1:5000 (Invitrogen).

Antibodies used were ZIP6-SC (E-20, SC-84875), pS⁷²⁷STAT3 (SC-8001-R and SC-136193), pY⁷⁰⁵STAT3 (SC-7993-R), total STAT3 (SC-8019), normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027) and GAPDH (SC-32233) from Santa Cruz Biotechnology; α-tubulin (DM1A, #3873S), pS¹⁰HistoneH3 (#9706S and #3377) and pS³⁸Stathmin (#4191) from Cell Signalling Technology; mouse V5 from Invitrogen; rabbit V5 (Ab9116), mouse/rabbit apoptosis cocktail (ab136812) from Abcam; β-actin (A5316), ZIP10 cytoplasmic loop antibody (SAB1401780) from Sigma-Aldrich (referred to as ZIP10S antibody). Treatments used were 100 ng/mL nocodazole (Sigma-Aldrich, M1404) for 20 h, 200 μM STAT3 inhibitor cell-permeable peptide (Calbiochem, 573,096), 5 μM camptothecin (Sigma- Aldrich) for 20 h and 20–100 μM zinc with 10 μM sodium pyrithione (Sigma-Aldrich), and 25 or 50 μM TPEN (Sigma-Aldrich). Mitosis inhibition experiments were performed using mouse monoclonal antibody generated by Biogenes to ZIP6 residues 240–253(ZIP6-Y) and rabbit polyclonal antibody to ZIP10 residues 46–59 (ZIP10B) which were added to cells in culture for 20 h in the presence of nocodazole.

For co-IP of overexpressed proteins, proteins were extracted from cells 24-48 h post transfection. For co-IP of endogenous LRRK2, SN4741 cells were lyzed in 90% of confluency 24 h after seeding. Mouse VM tissue was first frozen to −80 °C and lysed once needed. Cells were washed twice in ice-cold PBS, and lyzed in 1-2 ml of freshly prepared lysis buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.1 mM DTT and proteases inhibitors cocktail from Roche) on ice for 15 min. The lysis was followed by centrifugation of the lysates at 18000 g, +4 °C for 20 min to separate insoluble particles and cellular debris. To eliminate unspecific interactions, lysates were pre-cleared by incubating the lysates with DynaBeads (Invitrogen) for 45 min. To pull down the binding partners, 800 μl of lysates were incubated with 1 μg of following antibodies: goat GFP-FITC (Abcam), rabbit C-MYC (Sigma), mouse V5 (Invitrogene), rabbit FLAG (Sigma) and mouse LRRK2 (Covance), while rotating at +4 °C for 3 h. Afterwards, the protein complexes were incubated with DynaBeads for 12-14 h. The beads were washed 5 times using lysis buffer without DTT and proteases inhibitors. To elute the complexes from the beads, the beads were mixed with 1× Laemmli buffer and denaturized at 95 °C for 5 min. The beads were subsequently removed and samples loaded directly into 8-10% SDS-PAGE gel.