Anti β tubulin

Lysates of adult Drosophila heads were prepared using RIPA buffer supplemented with protease inhibitors (Sigma #11836170001). Antibodies used for immunoblotting with dilutions were: anti-dhtt (3526, rabbit polyclonal, 1:1,000; [13 (link)]), anti-dAbl (as above, 1:1,000; [58 (link)]), anti-β-Tubulin (mouse, DSHB E7, 1:10,000), anti-mouse and anti-rabbit IRDye secondary antibodies (LI-COR Biosciences, 1:10,000). Lysates were resolved on NuPAGE 3–8% gradient Tris-Acetate gels with Tris-Acetate running buffer (for Htt and Abl) or NuPAGE 4–12% gradient Bis-Tris gels with MOPS running buffer (for β-Tubulin). After transfer to nitrocellulose membranes, blots were processed according to the Odyssey CLx protocol. Median band intensity was quantified using Image Studio.

Whole cell protein extracts were washed with PBS and lysed with TNN lysis buffer with protease inhibitors. Purified protein extracts were then heated to 95 °C for 5 min and resolved through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After resolution, the gel was transferred to a nitrocellulose membrane (Whatman, Germany) for three hours at 250 mÅ at 4 °C in transfer buffer (25 mM Tris pH 7.4, 200 mM glycine, 20 % methanol). After transfer, membranes were blocked for thirty minutes at room temperature with 5 % non-fat dry milk in 1X phosphate-buffered saline containing 0.1 % Tween-20 (PBST). After blocking, membranes were washed and incubated with primary antibodies at a 1:1000 dilution overnight at 4 °C. After primary antibody incubation, membranes were washed three times in PBST and incubated with secondary antibodies at a 1:5000 dilution at room temperature. Following secondary antibody incubation, membranes were visualized with the Odyssey CLx Imaging System (LI-COR). Primary antibodies used were anti-T-antigen (pAb-416, Calbiochem), anti-SRSF1 (ab12910, Abcam), anti-agnoprotein (Pab7903, house raised), anti-β-Tubulin (LI-COR), and anti-GAPDH (Cell Signaling Technology). Secondary antibodies used were IRDye 800CW goat anti-mouse (LI-COR) and IRDye 680RD goat anti-rabbit (LI-COR).

The following antibodies were used in this study: anti-FLAG (WB 1:1,000, F3165; Sigma), anti-EPLIN (WB 1:1,000, IF 1:100, 50311; Cell Signaling Technology), anti-EPLIN (WB 1:1,000, IF 1:100, sc-136399; Santa Cruz Biotechnology), anti-EPLIN (WB 1:1,000, immunoprecipitation [IP] 1 μg/1 mg lysate, immunohistochemistry [IHC] 1:200, 16639–1-AP; Proteintech), anti-HA (WB 1:500, IP 2 µg/1 mg cell lysate, SC F-7; Santa Cruz Biotechnology), Alexa Fluor 568–phalloidin (IF 1:1,000, A1238; Life Technologies), SiR-actin (1:10,000, CY-SC001; Cytoskeleton), anti–β-tubulin (WB 1:2,500, 926–42211; LI-COR), anti–α-tubulin (WB 1:2,500, 23948; Santa Cruz Biotechnology), pFAK Y397 (WB 1:1,000, ab8129; Abcam), FAK S910 (WB 1:1,000, 44-596G; Invitrogen), anti–E-Cadherin (WB 1:1,000, IHC 1:200, 3195; Cell Signaling Technology), total FAK (WB 1:1,000, 610087; BD Biosciences), anti-zyxin (IF 1:1,000, ab50391; Abcam), anti-Rab40b (WB 1:500, LS-C353287; LSBio), anti-Rab40c (WB 1:500, H-8 sc-514826; Santa Cruz Biotechnology), and anti-CD63 (IF 1:100; gift from Andrew Peden, University of Shefield, Shefield, UK).