Rabbit anti α tubulin

Western blot assays were conducted as described previously [7 (link)]. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% or 10% gels (Thermo) and were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). PVDF membranes were blocked in 1 mg/mL BSA (Sigma-Aldrich) at room temperature for 1 h. Primary antibodies were diluted 1:2000 and incubated overnight at 4 °C. Secondary antibodies diluted 1:5000 were added and incubated at room temperature for 1 h. The band intensities corresponding to the protein samples were measured using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The primary antibodies used were as follows: mouse anti-β-catenin, mouse anti-lamin A/C (BD Biosciences, CA, USA), mouse anti-TCF4 clone 6H5-3 (Millipore), mouse anti-N-EBP1 (C11) (Santa Cruz, CA, USA), rabbit anti-EBP1, rabbit anti-histone H3, rabbit anti-α-tubulin, rabbit anti-HIF-1α and rabbit anti-β-actin (GeneTex, CA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology) were used as appropriate.

Transfected cells were washed twice with ice-cold PBS ((in mM): 136 NaCl, 2.5 KCl, 1.5 KH₂PO₄, and 6.5 Na₂HPO₄, pH 7.4), centrifuged, and resuspended in lysis buffer ((in mM): 150 NaCl, 5 EDTA, 50 Tris-HCl, 1 PMSF, 1% Triton X-100, pH 7.6, supplemented protease inhibitor mixture). Laemmli sample buffer was added to the lysates, and samples were sonicated on ice (3 times for 5 s each) and heated at 70 °C for 5 min. Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-Myc (1:5000; clone 9E10), rat anti-HA (1:5000; Roche), or rabbit anti-α-tubulin (1:5000; GeneTex). Blots were exposed to horseradish-peroxidase-conjugated goat anti-mouse IgG (1:5000; Jackson ImmunoResearch), goat anti-rabbit IgG (1:5000; Jackson ImmunoResearch), or goat anti-rat IgG (1:5000; Santa Cruz Biotechnology) and revealed by an enhanced chemiluminescence detection system (Thermo Scientific). Densitometric scans of immunoblots were quantified with ImageJ (National Institute of Health). Data shown are representative of at least three independent experiments.

For immunolabeling, embryos were fixed for 3 h with 4 % PFA diluted in MAB buffer (80mM KPIPES, 5mM EGTA, 1mM MgCl2, 0.2 % Triton-X, pH 6.4) at room temperature. Embryos were then sectioned (40μm, 1500 Sectioning System) and immunolabeling on floating sections was carried out as in [84 ].
Antibodies used: mouse anti-β-tubulin (Sigma, Clone: TUB 2.1) at 1:500; rabbit anti-α-tubulin (Genetex, Clone: GTX108784) at 1:500; rabbit anti-tyrosinated tubulin (Millipore, ABT171) at 1:1000; rabbit anti-glu-tubulin (Millipore, Clone: AB201) at 1:1000 and rabbit anti-GFP (Invitrogen, cat. no. A11122) at 1:1000. Secondary antibodies conjugated to Alexa 488, Alexa 594, or Cy3 (Molecular Probes, cat. nos A11001 and A11008; Molecular Probes cat. A21442; Invitrogen cat. no. A10520) were used at a 1:500 dilution. Alexa Fluor 488-conjugated Phalloidin (Invitrogen, cat. no. A12379) at 1:75 and DAPI (Invitrogen, cat. No. D1306) were used according to manufacturer’s instructions.
For cell shape analysis, mGFP or mRFP RNA/DNA injected embryos were sectioned and either imaged directly (mRFP) or immunolabeled with anti-GFP prior to imaging. All fluorescently labeled sections were imaged using an SP5 confocal microscope (Leica SP5 TCS 4D).