Rat anti ha 3f10

S2-DGRC cells (Cellosaurus CVCL_TZ72) were obtained from the Drosophila Genomics Resource Center and transfected with Effectene (Qiagen). For co-IP assays, cells were lysed in HEPES lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% (v/v) Triton X-100] supplemented with phosphatase inhibitor cocktails 1 and 2 (Sigma) and Protease Inhibitor Cocktail (Roche) on ice for 15 min. Soluble cell lysates were obtained after centrifugation at 15,000 g for 15 min at 4°C. Protein concentrations were determined using the Dc protein assay (Bio-Rad). Lysates were then incubated with Protein A/G sepharose beads and appropriate antibodies for 2 h at 4°C. Immunoprecipitates were then purified after washing four times with HEPES lysis buffer. Detection of purified proteins and associated complexes was performed by immunoblot analysis using chemiluminescence (GE Healthcare). Western blots were probed with anti-FLAG (mouse M2, Sigma; 1:1000), anti-Myc (mouse 9E10, Santa Cruz Biotechnology; 1:1000), anti-HA (rat 3F10, Roche; 1:1000) and anti-RASSF8 (Langton et al., 2009 (link)) antibodies.

Adult male dissected brains were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 25 minutes and labeled using a modification of protocols previously described [55 (link)]. The following primary antibodies were used: anti-bruchpilot (mAb nc82, 1:30, Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biology, University of Iowa (Iowa City, IA).), monoclonal rabbit anti-GFP (1:200, Molecular Probes), rat anti-HA 3F10 (1:100, Roche), mAb dVGLUT (1:15), anti-TβH (1:400, [89 (link)]), rat anti-V5 (1:200, Biorbyt), and rabbit anti-TDC2 (1:100, Covalab). Secondary antibodies conjugated to Alexa 488, Alexa 594, or Alexa 647 (Molecular Probes) were used at a concentration of 1:200. Labeled brains were mounted in Vectashield (Vector Labs, #H1000). Images were collected on an Olympus Fluoview FV1000 laser scanning confocal mounted on an inverted IX81 microscope and processed using ImageJ (NIH) and Adobe Photoshop (Adobe, CA).

Synchronized schizont cultures were magnet-purified and incubated in E64 (10 µM) for 3–5 hours until fully segmented. Parasites were passed through a 1.2 μm syringe filter to obtain merozoite suspension. Merozoites were mixed with erythrocytes in culture media supplemented with dimethyl sulfoxide or R1 peptide (100 µg/ml) and incubated at 37°C with shaking for 1 min 30 s until fixing. Parasite cultures were spun down at 2000×g and fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate-buffered saline (PBS) for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 25 min, and incubated in blocking solution (2% bovine serum albumin in PBS) for 1 hour at room temperature. Cell suspensions were then mounted onto 1% polyethyleneimine-treated coverslips, the excess liquid was removed and samples were mounted on slides with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI). The primary antibody used to detect the C-terminus of RON3 was rat anti-HA (3F10, Roche, 1:500) and the secondary antibody conjugated to Alexa-488 fluorophore from Thermofisher (1:1000). Wheat Germ Agglutinin 647 conjugate (Thermofisher) was used to stain the erythrocytes at 1:1000 dilution.

The following antibodies were used for western blotting: rat anti-HA (3F10; Roche/Sigma-Aldrich), rabbit anti-myc, mouse anti-RhoA, rabbit anti-MLC2 (Santa Cruz Technology), rabbit anti-FAM40B (Sigma-Aldrich), rabbit anti-pThr18/pSer19-MLC2 (Cell Signaling), mouse anti-GADPH (Millipore), secondary HRP-conjugated sheep anti-mouse IgG and donkey anti-rabbit IgG (GE Healthcare). Antibodies for immunofluorescence analysis were: rabbit anti-HA (Santa Cruz Technology), rabbit anti-pSer19-MLC2 (Cell Signaling), mouse anti-VE-cadherin (BD Biosciences), rabbit anti-ZO-1 (#61–7300; ThermoFisher Scientific), secondary AlexaFluor-488 and -647-conjugated antibodies (Molecular Probes). Cells were also stained with DAPI (DNA) and AlexaFluor-546-labeled phalloidin (F-actin; Molecular Probes).

Immunofluorescence of ovaries was performed as described with representative images shown [67 (link)]. Eye imaginal discs were dissected from 3rd instar larvae and immunostained as described with representative images shown [68 ]. Adult thoraxes were dissected and fixed in 4% paraformaldehyde/PBS at 4°C for 1 hour, and then cryoprotected in 12% sucrose/PBS at 4°C overnight before being embedded in OCT (Tissue Tek). Cryosections (10-μm thick) were immunostained as described [69 (link)]. Antibodies and dilutions used are: rat anti-HA 3F10 (1:200, Roche), mouse anti-ELAV-9F8A9 (1:10), rabbit anti-DmFRG1 DM1 (1:300), rabbit anti-DmFRG1 DM2 (1:300), and Alexa 594 or 488-conjugated secondary antibodies. Nuclei were visualized with DAPI (Invitrogen). Elav-9F8A9 was deposited to the Developmental Studies Hybridoma Bank by G.M. Rubin. For polytene chromosome spreads, salivary glands were dissected from >100 third-instar larvae grown at 25°C and immunofluorescence was performed as described [70 ].