Veriblot secondary antibody

Samples were resolved by SDS-PAGE (10–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated. Primary antibodies were used to detect Bcl-x_L (54H6, 1:1,000 dilution, Cell Signaling; 2H12, 1:500 dilution, Invitrogen), COX IV (3E11, 1:20,000 dilution, Cell Signaling), Drp1 (D6C7, 1:1,000 dilution, Cell Signaling), Flag (66008-3-Ig, 1:1,000 dilution, Proteintech), GAPDH (sc-365062, 1:1,000 dilution, Santa Cruz biotechnology), GFP (sc-8334, 1:1,000 dilution, Santa Cruz biotechnology), HA (51064-2-AP, 1:1,000 dilution, Proteintech), Mff (17090-1-AP, 1:2,000 dilution, Proteintech), PARP (46D11; 1:1,000 dilution, Cell Signaling), RFP (3F5, 1:1,000 dilution, ChromoTek), SENP3 (D20A10, 1:10,000 dilution, Cell Signaling), and β-actin (A2228, 1:20,000 dilution, Sigma). Immune complexes were detected either using HRP-conjugated secondary antibodies (Sigma) or an HRP-conjugated VeriBlot secondary antibody (ab131366, Abcam; for immunoblotting involving IP samples) followed by enhanced chemiluminescence (GE Healthcare Amersham), or using fluorescent secondary antibodies (Thermo Fisher Scientific) by a LI-COR imaging system. Each immunoblot presented is representative of at least three independent experiments carried out using different cell populations.

To assess endogenous LRRK2 S1292 phosphorylation in VPS35 wild-type, D620N heterozygous, and D620N homozygous MEFS, LRRK2 was immunoprecipitated from lysates (2 mg of protein) using 4 µg of anti-LRRK2 antibody UDD3 coupled to Protein A Sepharose beads. Immunoprecipitated LRRK2 was washed three times with lysis buffer and eluted from the beads with 2× NuPAGE LDS Sample Buffer. Eluted samples were used for detecting total LRRK2 and phospho-Ser1292 LRRK2 by immunoblotting analysis. For detecting immunoprecipitated LRRK2 (phospho and total), VeriBlot secondary antibody (Abcam, #ab131366) was used instead of normal anti-rabbit IgG secondary antibody.

Cell lysates were prepared with IP lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol). Buffer was supplemented with protease inhibitor cocktail (HALT, ThermoFisher). Lysates were sonicated and cleared by centrifugation. Lysates were incubated with 1 µg of antibody overnight and protein complexes precipitated using Protein A/G magnetic beads (ThermoFisher Scientific). Washes were performed using TBS buffer with 0.05% Tween-20. Proteins were eluted from beads by boiling with LDS sample buffer. Proteins were analyzed by western blot as described and probed using Veriblot secondary antibody (Abcam).