Mouse anti α tubulin dm1a

Goat anti-FE65 (E-20), mouse anti-ARF6 (3A-1), mouse anti-ARNO (H-7), mouse anti-c-Jun (D-11) and mouse anti-α-Tubulin (DM1A) were purchased from Santa Cruz. Rabbit anti-ARNO, rabbit anti-GFP, rabbit anti-His and rabbit anti-FLAG were obtained from Proteintech. Mouse anti-myc antibody (9B11) and rabbit anti-COX IV (3E11) were obtained from Cell Signaling Technology. Mouse anti-pan-cadherin (C1821), mouse anti-β-COP (maD) and mouse anti-FLAG antibody (M2) were obtained from Sigma. Goat anti-GST antibody was obtained from GeneTex. Mouse anti-GAPDH (AM4300) was purchased from Ambion. Rabbit anti-β-Tubulin was purchased from Abcam. Rabbit anti-FE65 was as previously described [13 (link),14 (link)]. Rat polyclonal antibodies against ARF6, ARNO and GST were created by immunization of rats with ARF6, ARNO and GST bacterial proteins, respectively.

Rabbit anti-FE65 and rabbit anti-APP A5137 were as described^{5 (link)}. Goat anti-FE65 E20 and mouse anti-α-tubulin DM1A were obtained from Santa Cruz. Mouse anti-myc 9B11, mouse anti-GFP JL8, mouse anti-HA 12CA5, rabbit anti-GST and mouse anti-APP 22C11 were purchased from Cell Signaling Technology, Clontech, Roche, Sigma and Millipore, respectively.
The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579. A cysteine (C) residue was introduced to the peptide C-terminus for carrier protein conjugation and purification column coupling. Phosphopeptide conjugation was performed using the Imject Maleimide Activated mcKLH Spin kit (ThermoFisher Scientific). The immunized rat sera were purified by the non-phosphopeptide (REQWTPSHVSVC) and then phosphopeptide coupled columns prepared by using a SulfoLink Immobilization kit for peptides (ThermoFisher Scientific). The antibody was eluted and dialyzed against PBS/0.05% sodium azide, and then concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore).

Ovaries were dissected in Grace’s medium and processed for IP as described previously [16 (link)]. For western blotting, one-half ovary equivalent lysate was loaded into each lane of an 8% or 10% SDS-PAGE. The following primary antibodies were used: mouse anti-Aub (1:1,000, from Dr Siomi), mouse anti-Ago3 (1:500, from Dr Siomi), rat anti-Tap (1:1,000, this study), mouse anti-c-Myc 9E10 (1:5000, Sigma), mouse anti-HA (1:5,000, Roche, BASEL, Switzerland), mouse anti-FLAG M2 and its horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000, Sigma), guinea pig anti-Vas (1:5,000) [16 (link)], rabbit anti-Piwi (1:500, Abcam, Cambridge, England, United Kingdom Ab5207), mouse anti-Piwi (1:50, from Dr Siomi), rabbit anti-SpnE (1:500, from Dr Dahua Chen) and mouse anti-α-Tubulin DM1A (1:1,000, Santa Cruz Biotechnology, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). Immunoreactive bands were visualized using HRP-conjugated goat anti-guinea pig (Dako, Dako North America, Inc. Carpinteria, CA, USA), anti-rabbit, anti-rat or anti-mouse secondary antibodies (Bio-Rad, Hercules, CA, United States of America) at 1:5,000, and developed with the SuperSignal West Pico Chemiluminescent Substrate detection reagent (Thermo Scientific).