Anti ha 3f10

To confirm exogenous protein expression, DSC neurons electroporated with Dcc or Robo3 cDNA and gfp were microdissected, dissociated (0.05% trypsin, 0.5 mM EDTA), and cultured for 24 hrs in PDL (poly-D-Lysine, 100 μg/ml) coated culture dish in the culture medium (Neurobasal, 1x B27, 50 U/ml Pen/Strep, and 250 ng/ml Netrin-1). Cells were then fixed with 4% paraformaldehyde, and stained with anti-HA (3F10, Roche) and Alexa Fluor 594-conjugated secondary antibodies (Jackson ImmunoResearch).

FBXW7 substrates conjugated with Myc tag (cyclin E and BRAF) were analyzed by anti-Myc (9E10; Roche), whereas anti-HA (3F10) (Roche) was used to detect FBXW7 substrates conjugated with HA tag (c-MYC and MCL1). Anti-Flag M2(Sigma #F3165) was used to for FBXW7 and mTOR (Addgene #26603) detection. Endogenous expression of proteins was performed using antibodies for CyclinE (Santa-Cruz #sc-198), Mcl-1 (Santa-Cruz #sc-12,756), c-myc (9E10), FBXW7 (Proteintech #28424–1-AP), BRAF (Santa-cruz #sc-5284), p-MEK-1/2 (Calbiochem), p-ERK-1/2, and STAT3 (Santa-Cruz #sc-482). All western blots were normalized to Actin expression (Santa-Cruz #sc-8432). For co-immunoprecipitations, 293 T cells were co-transfected with tagged FBXW7 and its substrate(s) for 48 h. Cells were lysed in NP-40 lysis buffer (50 mM KCl, 10 mM Tris-HCl pH 8.8, 5 mM MgCl2 and 0.65% NP-40), immunoprecipitated with appropriate tagged antibodies, and immunoblotted by the indicated antibody as described in the figure legends.

Immune detection of epitope-tagged Kti12 and Elongator subunits and immune precipitations (IPs) from total yeast extracts used anti-c-Myc (9E10) and anti-HA (3F10) antibodies (Roche) as previously described [6 (link),24 (link)]. Protein loading was checked with anti-Cdc19 serum (a kind donation by Dr J. Thorner, University of California, Berkley, CA, USA), which recognizes pyruvate kinase. CaM binding assays in vitro involved TAP-tagged or c-Myc-marked Kti12 and Kti13 proteins from total yeast extracts (~2 mg) adjusted to 1 mM CaCl₂. The material (2 mL) was mixed with CaM-Sepharose for 30 min and equilibrated with 2 mL CaM binding buffer (CBB) as described [32 (link)]. The slurry was packed onto a column, the buffer drained, and the column rinsed twice with 2.5 volumes of CBB. Elution of bound proteins was in EGTA buffer (4 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM MgCl₂, 2 mM EGTA, and 0.1 mM DTT). Equal proportions of load, flow-through, wash, and elution fractions were separated by 15% SDS-PAGE, and Kti12 material was detected using anti-TAP and anti-c-Myc Western blots.

Culturing of chicken embryos and in ovo electroporation were performed as described previously (Chen et al., 2008 (link)). Actb-gfp (βactin-gfp) and Robo1/2 expression constructs (in the pCAGGS vector) were microinjected into the neural tube of E3 chicken embryos (stage 18) and electroporated into one half of the spinal cord (ECM830 electroporator, 30 volts, 50 ms/pulse, five pulses). The concentration of the plasmids was 150 ng/μl. After culturing for an additional 48 hr at 37°C (until stage 25), spinal cords were microdissected, fixed with 4% PFA, and imaged in an openbook configuration with fluorescence microscopy. Commissural axons were distinguished from ipsilateral axons as the former turned longitudinally next to the midline while the latter turned at a more dorsolateral position. To assess exogenously expressed mouse ROBO proteins, anti-HA (3F10, Roche) was used to stain transverse sections of chicken spinal cord. In addition, we lysed chicken spinal cords with lysis buffer (10 mM Tris, 150 mM NaCl, 1% Triton X-100, pH 8, with protease inhibitors). After centrifugation to remove undissolved materials, SDS loading buffer (4x with 200 mM Tris-HCl pH6.8, 8% SDS, 0.4% bromophenol blue, 400 mM DTT) was added to the supernatant and the lysate was analyzed by SDS-PAGE using anti-HA conjugated with HRP (Roche).