Rabbit anti kif5b

HEK 293T cells or DRG sensory neurons were collected and prepared with lysis buffer (1% NP-40; 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; protease inhibitor [Sigma-Aldrich]; and phosphatase inhibitor [Life Technologies]). Cell lysates were placed on ice for 20 min and centrifuged at 13,000 rpm for 20 min to collect the supernatant. Lysates were separated with 4–12% Bis-Tris NuPAGE gel (Thermo Fisher Scientific) and blotted with the following primary antibodies: mouse anti-SFPQ (1:1,000; Sigma-Aldrich), rabbit anti-KIF5A (1:2,000; Abcam), rabbit anti-KIF5B (1:2,000; Abcam), rabbit anti-KIF5C (1:2,000; Abcam), rabbit anti-KIF3C (1:500; Abcam), rabbit anti-KIF3A (1:200; Abcam), rabbit anti-KIF1A (1:5,000; Abcam), mouse anti-HAlo (1:1,000), rabbit anti-KLC1 (1:500; Santa Cruz Biotechnology), rabbit anti-KLC2 (1:1,000; Proteintech), rabbit anti-pan actin (1:1,000; Cell Signaling Technologies), mouse anti-Myc (1:500; Santa Cruz Biotechnology), mouse anti-FLAG (1:1,000; Sigma-Aldrich), mouse anti-HA (1:10,000; Thermo Fisher Scientific), and rabbit anti-GFP (1:2m000; Sigma-Aldrich). HRP-conjugated secondary antibodies (1:10,000; BioRad), ECL detection system (VWR International), and SuperSignal West Dura (Thermo Fisher Scientific) were used for chemiluminescent detection.

The RPE-choroid was isolated from WT and Klc1^−/− mouse eyes and lysed in 20 mM Tris, pH 7.4, 5 mM MgCl₂, 10 mM NaCl, 1 mM DTT, and 1× protease inhibitors (Sigma-Aldrich). Equivalent amounts of sample were run on a 4–12% NuPAGE Bis-Tris gel (Invitrogen). After transfer, membranes were blocked with Odyssey blocking buffer (LI-COR; Lincoln) and probed with rabbit anti-calnexin (Enzo Life Sciences), rabbit anti-KIF5B (Abcam), rabbit anti-KLC1 (Santa Cruz Biotechnology, Inc.), and mouse anti-GAPDH (EMD Millipore). Goat anti–mouse IRDye 680, donkey anti–rabbit 680, or donkey anti–rabbit 800 secondary antibody (LI-COR; Lincoln) was used. Membranes were imaged with the Odyssey infrared imaging system (LI-COR; Lincoln).