Mouse α gfp

Twenty microgram of protein from whole‐cell lysate or 15 μL of eluted proteins from immunoprecipitation were separated by Tris/glycine SDS/PAGE (8% α‐actinin, 10% tubulin or actin, 12% SIKE), and transferred to a nitrocellulose membrane. The membrane was blocked in either 5% milk or 5% BSA (rabbit α‐tubulin‐HRP) in TBS/Tween. The immunoblot was probed for SIKE using mouse anti‐FLAG‐HRP or mouse anti‐HA or rabbit anti‐SIKE or cytoskeletal protein using either mouse‐α‐GFP (BioLegend) or rabbit α‐tubulin‐HRP (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The blot was then washed with TBS/Tween: 6 × 5 min (rabbit anti‐FLAG‐HRP) or 3 × 5 min (all other blots). For blots requiring a secondary antibody, goat anti‐mouse or goat anti‐rabbit IgG‐HRP (Southern Biotech, Birmingham, AL, USA), was incubated with blot for 1 h, and washed with TBS/Tween (4 × 10 min). See Table 2 for antibody dilutions. Membranes were developed with Pierce ECL Plus reagent (Thermo Fisher Scientific) as per manufacturer's protocol and documented on a Gel Doc XR+ gel documentation system (Bio‐Rad) or a c‐DiGit blot scanner (LI‐COR, Lincoln, NE, USA). For whole lysate immunoblots, the blots were stripped, blocked in 5% milk TBS/Tween, probed for actin using mouse anti‐actin‐HRP, washed with TBS/Tween (7 × 5 min), and developed as described above.