Anti ha y 11

Cells grown on 13 mm coverslips were fixed in 4% paraformaldehyde (w/v) in PBS for 15 min, prior to permeabilisation in 0.5% Triton X-100 (v/v) in PBS for 15 min. Cells were then blocked in blocking buffer for 1 h, prior to 1 h labelling with primary antibody at 1 : 500 dilution (anti-HA (Y-11) (Santa Cruz) or anti-Flag antibody). Cells were washed three times before being stained with secondary antibody for 1 h (anti-mouse and anti-rabbit Alexa Fluor 488 and 594 antibodies (Thermo Fisher)). Cells were washed three more times and then mounted on glass slides using Vectorshield mounting media containing DAPI (Vectorlabs; Peterborough, UK). Images were collected using Openlab5 software (Improvision; Coventry, UK) and an Olympus BX60 fluorescent microscope fitted with a Hamamatsu C4742-95 camera.

Total protein lysate was quantified using a standard Bradford assay and 10 μg of lysate was used for immunoblotting experiments. For Blue Native PAGE, cells were lysed in 1x Sample Preparation buffer (ThermoFisher) containing 1% digitonin. 1% SDS was supplemented for SDS-PAGE. All proteins were transferred to PVDF membranes using TurboBlot (Bio-rad) at 2.5 mA for seven mins. The primary antibodies used were anti-FLAG (M2, Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GAPDH (Santa Cruz Biotechnology Cat# sc-47724, RRID:AB_627678) and anti-HA (Y-11, Santa Cruz Biotechnology Cat# sc-805, RRID:AB_631618). The secondary antibodies were anti-rabbit IgG-HRP (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567), anti-mouse IgG-HRP (light chain specific) (Jackson ImmunoResearch Labs Cat# 205-032-176, RRID:AB_2339056) and anti-mouse IgG-HRP (Jackson ImmunoResearch Labs Cat# 115-035-003, RRID:AB_10015289).

COS cells were transfected with 1 μg of pShuttle-IRES-hrGFP-2 expressing either empty vector, WT-EphB4-HA, or mutant-EphB4-HA. The mutant-EphB4-HA plasmids consisted of: Y574F-, Y581F-, Y590F-, Y596F-, Y614F-, Y653F-, Y730F-, Y736F-, Y774F-, Y806F-, Y821F-, Y837F-, Y906F-, or Y924F-substitution. After 24 hr starvation, the transfected Cos-7 cells were treated with Ephrin-B2/Fc (2 μg/ml) for 1 min. Cell lysates were then harvested and prepared. Whole cell lysates were then immunoprecipitated for the HA-tag. The IP sample underwent immunoblotting for both HA (Eph-B4) and pTyr using the primary antibodies anti-HA [Y-11] (Santa Cruz, #sc-805) and anti-pTyr [4G10] (Millipore, #05-321), respectively.

HEK293 cells were transfected by lipofectamine (Invitrogen) and then lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA [pH 8.0], 0.5% sodium deoxylcholate, 10 mM phenylmethylsulfonyl fluoride, and 1M dithiothreitol) with 1% NP-40. For co-immunoprecipitation, cell lysates were immunoprecipitated by anti-HA (Invitrogen) or anti-FLAG (Sigma) beads. Immuno-complexes or samples for western blot analysis were electrophoresed in a SDS-polyacryalamide gel, transferred onto PVDF membrane and probed with anti-HA Y-11(1:4000, Santa Cruz) or anti-FLAG (1:5000, Sigma) antibodies.

SDS-PAGE and immunoblotting were performed with standard protocols described previously using 5 to 10% acrylamide gels [57 (link)]. SDS-PAGE gels were stained with CBB (Coomassie brilliant blue) or silver. For immunoblotting, samples separated by SDS-PAGE were transferred to a nitrocellulose or PVDF membrane, stained with CBB or Ponceau S if necessary, incubated with specific primary antibodies and subsequently HRP (horseradish peroxidase)-conjugated secondary antibodies. Immuno-reaction was detected using a TMB (3,3′,5,5′-tetramethylbenzidine peroxidase) substrate kit (Vector Laboratories), Pierce ECL immnoblotting substrate (Thermo Scientific) or ECL prime immunoblotting detection reagent (GE Healthcare). Primary antibodies used were as follows: anti-DYX1C1 CT299 (Rabbit: this study), anti-PF13/KTU (Rabbit: [17 (link)]), anti-IDA10/MOT48 (Rabbit: this study), anti-IC138 (Rabbit: [62 (link)]), anti-IC2 (Mouse: [63 (link)]), anti-Actin (Rabbit: [64 (link)]), anti-p28 (Rabbit: [59 (link)]), and anti-HA 3F10 (Rat: Roche Applied Science) or anti-HA Y11 (Rabbit: Santa Cruz). HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies were commercially purchased from Invitrogen. Anti-PF13/KTU antibody was a generous gift from Dr. David R. Mitchell (SUNY Upstate Medical University).

10⁴ cells were seeded onto an 8-well Lab-TekII chamber slide, fixed in 3.7% formaldehyde solution (Sigma), and permeabilized in 0.1% Triton 100X (Sigma), PBS buffer at room temperature for 10 min. The cells were then washed 3 times with PBS and incubated for 1 h with 10% horse serum in PBS blocking solution. The cells were incubated overnight at 4°C with murine monoclonal anti-E-cadherin clone 36, (Becton Dickinson), anti-β-catenin clone 14 (Becton Dickinson), anti-fibronectin clone 10 (Becton Dickinson), anti-vimentin clone V9 (Dako), or with a polyclonal rabbit anti-HA Y11 (Santa-Cruz) primary antibody, washed in PBS, and then incubated for 1 h at room temperature with a goat anti-mouse Alexa Fluor 533 secondary antibody (A21422, Invitrogen) or with a mouse anti-rabbit Alexa Fluor 555 secondary antidody (A21428, Invitrogen). After extensive washes in PBS, the nuclei were stained with 5 mg/ml Hoechst for 10 min and mounted with Fluoromount-G (SouthernBiotech). All matched samples were photographed with an immunofluorescence microscope (Leica) and identical exposure times.