Anti kinesin

pCDNA-V5-mSmo was generated by introducing DNA sequence encoding the V5 epitope tag (GKPIPNPLLGLDST) after amino acid 51 in pCDNA-mSmo [36 (link)] using Phusion site directed mutagenesis kit (Thermo Scientific). Asn (N) to Gln (Q) mutagenesis was performed on Drosophila and mouse Smo expression vectors using QuikChange II Mutagenesis Kit (Stratagene). dBip, dCnx and dCrc expression plasmids were generated by cloning the respective cDNAs obtained from the DGRC into pAc5.1 (Life technologies). pCS2-YFP-mSmo, the pAc-Cal-KDEL ER marker and pAc-Myc-dSmo were described previously [10 (link),38 (link),70 (link)].
Antibodies used for western blot analyses include anti-mSmo (SCBT), anti-GFP (Cell Signaling), anti-V5 (Life technologies), anti-kinesin (Cytoskeleton, Inc.), anti-alpha tubulin (cell signaling) and anti-Myc (Sigma) and anti-SmoC [15 (link)].

Flies were dissected in Grace's media and ovaries were fixed and stained as described previously (Tanner et al., 2011 (link)). Samples were mounted in VectaShield with DAPI (Vector Labs). Primary antibodies used were: anti-cleaved-Dcp-1 (1:100, Cell Signaling Technology), anti-Dlg [1:100, Developmental Studies Hybridoma Bank (DSHB)], anti-αPS3 (1:300, Shigeo Hayashi, or 1:1000), anti-βPS (1:10, DSHB), anti-Drpr (1:50, DSHB 5D14), anti-β-Gal (1:400, Promega), anti-aPKC (1:1000, Santa Cruz Biotechnology, Inc.), anti-Dynein (1:3, DSHB 2C11), anti-Kinesin (1:100, Cytoskeleton, Inc.), anti-Crumbs (1:25, DSHB Cq4, protocol from Tanentzapf et al., 2000 (link)) and anti-Talin (mixture of A22A and E16B, 1:50 each, DSHB). The anti-αPS3 antibody was made using the peptide sequence utilized for the original antibody (Wada et al., 2007 (link)) and generated by YenZym (San Francisco, CA). The serum was affinity-purified twice before use. It shows the same expression pattern as the original antibody from the Hayashi lab. Secondary antibodies used were goat-anti-rabbit Cy3, goat-anti-mouse Cy3, goat-anti-mouse Alexa Fluor 647 (Jackson ImmunoResearch), each at 1:100, and goat-anti-rabbit Alexa Fluor 488 (Invitrogen) at 1:200. Egg chambers were imaged on an Olympus FV10i confocal microscope, images were processed using ImageJ and Adobe Photoshop, and figures were made using Adobe Illustrator.