Rabbit anti epha7

Total protein was isolated from cultured MDA MB 231, MDA MB 468, and MDAMB453 TNBC cells as well as from cultured MCF10A fibrocystic disease cells with the lysis buffer RIPA plus protease inhibitor cocktail. The homogenates were centrifuged at 17,000× g for 15 min at 4 °C. The supernatants were collected, and protein concentration was measured with the Bradford protein assay (Bio-Rad protein assay, Hercules, CA, USA). Thirty micrograms of protein samples were loaded each time into SDS-PAGE gels and transferred to nitrocellulose membranes (Whatman, Little Chalfont, UK) using the semi-dry transfer system (Bio-Rad). The membranes were blocked with 5% dry milk dissolved in Tris-buffered saline (1×) containing 0.1% Tween-20 for 1 h at room temperature (RT). The membranes were incubated with primary antibodies at 4 °C overnight followed by secondary antibodies for 1.30 h at RT. The primary antibodies in the Western blot were rabbit anti-EPHA2 (Santa Cruz, CA, USA, sc-924), mouse anti-EPHA4 (SantaCruz, sc-D-4), rabbit anti-EPHA7 (Santa Cruz, sc-1015), and mouse anti-beta actin (Sigma, A5441). The secondary antibodies were rabbit anti-mouse IgG (Sigma, A9044) (1:20,000 dilution) and goat anti-rabbit IgG (Sigma, A6154) (1:10,000 dilution).

Cell lysates were collected as described and denatured by boiling in Laemmli sample buffer with DTT before SDS-PAGE separation on 12% pre-cast gels (Bio-rad). Western blots were performed using the Bio-Rad Mini TransBlot and TransBlot-Turbo Transfer System, with the Bio-Rad PVDF Transfer Kit. Chemiluminescent imaging was performed on the ImageQuant LAS4000 biomolecular imager (GE Healthcare). The following primary antibodies were used: rabbit anti-EphA7 (Santa Cruz, 1:500), mouse anti-HA (Sigma, 1:1000), rabbit anti-HA (CST, 1:1000), mouse anti-myc (Sigma, 1:1000), rabbit anti-myc (CST, 1:1000), mouse anti-actin (Sigma, 1:2500), rabbit anti-phospho-tyrosine (CST, 1:1000), mouse anti-psd95 (CST, 1:1000), rabbit anti-Akt(pan) (CST, 1:1000). Appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were used at a dilution of 1:2500. When appropriate, bands were quantified via densitometry with GelQuant software (BiochemLab Solutions), averaged from two experiments, and compared using one- or two-way ANOVA with Bonferroni’s multiple comparisons correction (Graphpad Prism 6).

The protocol for immunohistochemistry has been described previously [20 (link),21 (link)]. The following primary antibodies were used for fluorescent immunohistochemistry on cryosections: rabbit anti-En1 (1:5000), rabbit anti-Lim1 (kindly provided by T.M. Jessell), rabbit anti-EphA4 (1:500, Santa Cruz), rabbit anti-EphA7 (1:100, Santa Cruz), mouse anti-Isl1 39.4D5 (1:50), and mouse anti-Neurofilament 3H5 (1:50, obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242). Antibody staining was visualized using fluorochrome-conjugated secondary antibodies (1:250; Molecular Probes; Jackson Dianova).