Goat secondary antibodies coupled to hrp

Samples were resolved by 10 or 12% SDS-PAGE and transferred to 0.2-µm nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris, 140 mM NaCl, and 0.1% Tween, pH 7.6) and probed at 4°C overnight with the following primary antibodies: rabbit anti-KNTC1 GC7, 1:3,000; mouse anti–α-tubulin B512 (Sigma-Aldrich), 1:5,000; rabbit anti-mCherry OD78, 1:17,000; rabbit anti–ZWL-1 OD89, 1:1,000; rabbit anti–SPDL-1 OD164, 1:1,000; rabbit anti-GST GC3, 1:7,500; mouse anti–6×His His.H8 (EMD Millipore), 1:2,500; mouse anti-p150 (BD), 1:500; mouse anti-dynein IC 70.1 (Sigma-Aldrich), 1:500; rabbit anti–DNC-1 GC2, 1:500; and rabbit anti–DNC-2 GC5, 1:5,000. Membranes were washed three times with TBST, incubated with goat secondary antibodies coupled to HRP (Jackson ImmunoResearch Laboratories, Inc.; 1:10,000) for 1 h at room temperature, and washed again three times with TBST. Blots were visualized by chemiluminescence using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and x-ray film (Fujifilm). For Strep-Tactin detection, membranes were blocked with 3% BSA in PBS/0.5% Tween 20, washed three times with PBS/0.1% Tween 20, and probed at 4°C overnight with Strep-Tactin HRP conjugate.