Rabbit anti v5

The following antibodies and reagents were used in this study: mouse anti-Flag M2 (1:5000, Sigma), rabbit anti-V5 (1:5000, Sigma), mouse anti-His (1:4000, Beyotime), mouse anti-HA (1:5000, Sigma), mouse monoclonal anti-dorsal (AB_528204, Developmental Studies Hybridoma Bank), mouse anti-Tubulin (1:6000, Vazyme), anti-FLAG M2 agarose beads (Sigma), mouse anti-Lamin (ADL67.10), anti-V5 agarose beads (Sigma), anti-HA-agarose beads (Sigma), goat anti-rabbit IgG-HRP (1:10000, Sigma), and goat anti-mouse IgG-HRP (1:10000, Sigma). A commercial antibody against Drosophila Relish (Abin1111036, RayBiotech 130–10080) was applied for immuno-staining [41 (link),42 (link)] and a polyclonal antiserum against Drosophila Relish [40 (link)] was applied to a Western blot to detect Relish in w¹¹¹⁸ and His2Av⁸¹⁰ mutants.

Antibodies used for immunoprecipitation and immunolabeling include rabbit anti-BIN1 exon 13 (custom made, generous gift from Sarcotein), mouse anti-BIN1 exon 17 (clone 99D, Sigma), recombinant monoclonal anti-BIN1 exon 13 (generous gift from Sarcotein), chicken anti-GFP (Abcam), mouse anti-Flag (Sigma), rabbit anti-CHMP4B (Abcam), rabbit anti-V5 (Sigma), rabbit anti-actin (Sigma), mouse and rabbit anti-GST (Santa Cruz), mouse anti-CD63 (Thermo Scientifics), fluorescein-conjugated anti-PI(4,5)P2 IgM (Echelon Bioscience), and fluorescein-conjugated anti-PI(3,4,5)P3 IgM (Echelon Bioscience). Alexa 647-conjugated WGA, Alexa 488-conjugated annexin V, Alexa 555-conjugated annexin V, Alexa 647-conjugated annexin V, and Alexa 647-conjugated phalloidin were purchased from Life Technologies. For imaging and flow cytometry, recombinant anti-BIN1 exon 13 was conjugated with Alexa 647 using a monoclonal antibody labeling kit (Life Technologies).

Fixation and antibody staining in imaginal discs were performed as described [70 (link)]. Fixation and antibody staining in cultured cells were performed as described [71 (link)]. Fixation and antibody staining in midguts were performed as described [37 (link)]. Primary antibodies used for the immunostaining were: mouse anti-Wdp (1:1000), chicken anti-lacZ (Abcam, 1:1000), mouse anti-Dl (DSHB, 1:50), mouse anti-Pros (DSHB, 1:200), rabbit anti-PH3 (Millipore, 1: 2000), mouse anti Brdu (DSHB, 1:200), rabbit anti Pdm1(1:1000, gift from Xiaohang Yang), mouse anti-V5 (Invitrogen, 1:3000), mouse anti-HA (Abmart, 1:500), rabbit anti-GM130 (Abcam, 1:200), rabbit anti-Rab5 (Abcam, 1:200), Gp anti-Sens (1:200), rabbit anti-Sal (1:100), and Rat anti-Ci (DSHB, 1:5). The primary antibodies were detected by fluorescent-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories, Inc. The primary antibodies used for IP and western blot were: rabbit anti-V5 (Sigma, 1:1000), rabbit anti-HA (Santa Cruz, 1:1000), mouse anti-Wdp (1:500), rabbit anti-GFP (Abmart, 1:1000) and mouse anti-tubulin (Abmart, 1:1000).

Dissected Drosophila guts were fixed at room temperature for 20 min in PBS, 4% paraformaldehyde. All subsequent incubations were done in PBS, 4% horse serum, 0.2% Triton X‐100 at 4°C following standard protocols.
The following primary antibodies were used at the following dilutions: guinea pig anti‐Ret 1/1,000 (Soba et al, 2015), mouse anti‐Pdm1 1/20 [kind gift from Steve Cohen, generated by Yeo et al (1995)], mouse anti‐Wg 1/20 (4D4 Developmental Studies Hybridoma Bank), mouse anti‐Arm 1/100 (N2 7A1 Developmental Studies Hybridoma Bank), mouse anti‐Pros 1/50 (MR1A, Developmental Studies Hybridoma Bank), chicken anti‐beta galactosidase 1/200 (ab9361, Abcam), goat anti‐HRP 1/600 (123‐095‐021 Jackson ImmunoResearch), rabbit anti‐p‐Src 1/100 (44660G; Invitrogen), rabbit anti‐pH3 (Ser10) 1/500 (9701L, Cell Signalling Technology), mouse anti‐GFP 1/1,000 (11814460001, Roche), rabbit anti‐V5 1/200 (V8137, Sigma) and rabbit anti‐phospho‐FAK (Tyr397) 1/250 (3283, Cell Signalling Technology).
Fluorescent secondary antibodies (FITC‐, Cy3‐ and Cy5‐conjugated) were obtained from Jackson Immunoresearch. Vectashield with DAPI (Vector Labs) was used to stain DNA.