Rabbit anti pkcζ c 20

Dechorionated embryos were homogenized in ice-cold lysis buffer (1% Triton X-100, 50 mM TRIS HCl pH 7.5, 5% glycerol, 100 mM NaCl, 50 mM NaF, 5 mM EDTA pH 8, 40 mM β-glycerophosphate, 1 mM PMSF, 0.5 μg/mL aprotinin, 0.7 μg/mL pepstatin, 0.5 μg/mL leupeptin and 0.1 mM orthovanadate) and processed for SDS-PAGE and western blotting as previously described (Laprise et al., 2002). Primary antibodies used were: guinea pig anti-Girdin 163 [3 (link)], 1:2,000; rabbit anti-Yrt (this study), 1:5000; mouse anti-Gapdh1 (Medimabs), 1:500; rabbit anti-Lgl D300 (Santa Cruz Biotechnology) 1:1,000; rabbit anti-PKCζ C20 (Santa Cruz Biotechnology), 1:2,000; mouse anti-Actin (Novus Biologicals), 1:10,00; mouse anti- β−Tubulin (DM1A, Sigma), 1:10,000; rabbit anti-GIRDIN (ABT80, Millipore), 1:1,000. HRP-conjugated secondary antibodies were used at a 1:2,000 to 1:10,000 dilution.

Ovaries and imaginal discs were dissected in PBS, fixed for 20 min in 4% paraformaldehyde in PBS, washed for 30 min in PBS/0.1% Triton X-100 (PBST) and blocked for 15 min in 5% normal goat serum/PBST (PBST/NGS). Primary antibodies were diluted in PBST/NGS and samples were incubated overnight at 4°C.
Primary antibodies used were: mouse anti-Cut (1:100, DSHB), mouse anti-β-gal (Promega, 1:500), rabbit anti-expanded (1:200, a gift from A. Laughon, University of Wisconsin-Madison, USA), rabbit anti-PKCζ (C-20) (1:250, Santa Cruz), rat anti-Crumbs (1:300, a gift from U. Tepass, University of Toronto, Canada), mouse anti-Dlg (1:250, DSHB), rabbit anti-Kibra (1:200; Genevet et al., 2010 (link)), rabbit anti-Src pY418 (1:150, Life Tech), FITC-conjugated anti-GFP (1:400, Abcam), rabbit anti-βH-Spectrin (1:100, a gift from G. Thomas, Pennsylvania State University, USA), guinea pig anti-Merlin (1:100, a gift from R. Fehon, University of Chicago, IL, USA), mouse anti-eyes absent (1:250, DSHB) and mouse anti-Coracle (1:100).
Secondary antibodies (all from Molecular Probes or Invitrogen) were used at 1:500 for 2-4 h prior to multiple washes in PBST and staining with DAPI at 1 mg/ml for 10-30 min prior to mounting on slides in Vectashield (Vector labs).

Ovaries and imaginal discs were dissected in PBS, fixed for 20 min in 4% paraformaldehyde in PBS, washed for 30 min in PBS/0.1% Triton X-100 (PBST) and blocked for 15 min in 5% normal goat serum/PBST (PBST/NGS). Primary antibodies were diluted in PBST/NGS, and samples were incubated overnight at 4°C. Optical cross sections through the middle of egg chambers are shown in all figures.
Primary antibodies used were as follows: mouse anti-Cut (1:100 DSHB), mouse anti-βgal (Promega, 1:500), rabbit anti-expanded (1:200, gift from A. Laughon, University of Wisconsin-Madison, Madison, WI, USA), rabbit anti-PKCζ (C-20) (1:250; Santa Cruz), rat anti-Crumbs (1:300; U. Tepass), mouse anti-Dlg (1:250, DSHB), rabbit anti-Kibra (1:200; Genevet et al, 2010 (link)) and guinea pig anti-Sec 15 (1:1,000, gift from H. Bellen, Howard Hughes Medical Institute, Baylor College of Medicine).
Secondary antibodies (all from Molecular Probes; Invitrogen) were used at 1:500 for 2–4 h prior to multiple washes in PBST and staining with DAPI at 1 μg/ml for 10–30 min prior to mounting on slides in Vectashield (Vector labs). Images were taken with a Leica SP5 confocal microscope and processed with Adobe Photoshop.