Anti alpha tubulin antibody

To extract proteins from primary, K4D, and K4DT + T cells, we lysed cells in a buffer containing 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 2.5 mg/ml sodium deoxycholate (Wako, Osaka, Japan; cat. no. 194-08311), and a protease inhibitor cocktail (1/200 dilution, Nacalai Tesque; cat. no. 25955-11). The protein expression levels of CDK4 and Cyclin D were detected by Western blotting using an anti-CDK4 antibody (1/2,500 dilution, MBL, Nagoya, Japan; cat. no. 25955-11), an anti-cyclin D antibody (1/5,000 dilution, MB; cat. no. 553), and an anti-alpha-tubulin antibody (1/1,000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-32,293). Anti-mouse IgG (1/2,000 dilution, GE Healthcare, Buckinghamshire, UK; cat. no. NA931) or anti-rabbit IgG (1/2,000 dilution; GE Healthcare; cat. no. NA934-1ML) was used as secondary antibodies. The detailed Western blot procedure has been reported previously (Fukuda et al., 2005 (link), 2008 (link)). The intensity of signals was measured by the Image J software.

Pools of 2 embryos at E18.5 were used for each of the three biological replicate. Total protein lysates were prepared by pulverizing skin tissue and extracted using RIPA (Boston Bio-Products) lysis buffer with protease inhibitor mixture (Roche). After Bradford protein assay (BIORAD), fifty micrograms subjected to 10% SDS–PAGE, transferred to nitrocellulose membranes (Schleicher and Schuell) and incubated with the following primary antibodies: anti-filaggrin (Covance), anti-loricrin (Covance) and anti-alpha tubulin antibody (Santa Cruz Biotechnology, Inc).
Mouse and rabbit secondary antibodies were used and the specific protein bands were detected using the ECL Plus kit (Pierce) and autoradiographic film (Hyperfilm ECL; Amersham Bioscience).
Statistical analysis was performed using unpaired t-test with significance evaluated by p value <0.05 (*) or <0.01 (**).

B16F10-Nex2 cells (4 × 10⁴) were seeded in round coverslips and incubated with 1.5 nmoles/10³ cells of biotinylated C36L1 and SC36 peptides for 30 min at 37 °C. Methanol was used for cell fixation for 10 min at room temperature. Cells were permeabilized in 0.1% Triton X-100, blocked with gelatin 2% at 37 °C for 10 min, washed in PBS and incubated with a secondary solution (10 μg/ml DAPI and 1:200 streptavidin FITC in water) for 15 minutes at 37 °C. To determine colocalization with microtubule cytoskeleton, the same protocol was carried out with primary anti-alpha-tubulin antibody (Santa Cruz, Biotecnology, Inc) incubated after cell permeabilization. Secondary anti-IgG PE antibody (Sigma) was used at 1:500 v/v. Images were taken in a Carl Zeiss LSM780 confocal microscope (magnification, ×1000).