Alpha tubulin

For sampling mouse tissues, dissected tissues were crushed using a homogenizer (μT-12, Taitec) in chilled RIPA buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The lysates were centrifuged at 20,400×g for 10 min to remove debris. Laemmli sample buffer was added to the lysates, followed by boiling at 95°C for 2 min. The protein samples (10 μg per lane) were separated by SDS-PAGE and transferred to a Hybond-P PVDF membrane (GE Healthcare). Because an antibody reactive to mouse Bex1 protein was not commercially available, we developed a new antibody reactive to mouse Bex1. Polyclonal antisera were generated by immunizing rabbits with the peptide [NH₂-C+KKEEKEEKPQDTIR-COOH] derived from the Bex1-specific amino acid sequence. The N terminal cysteine [C+] of the peptide was added for the purpose of conjugating the peptide to carrier protein. As loading control, alpha-tubulin (1:4000, Cell Signaling Technology, #3873) and Gapdh (1:4000, Cell Signaling Technology, #5174) were used. Secondary antibodies conjugated with horseradish peroxidase (Thermo Fisher Scientific, anti-mouse 32430 and anti-rabbit 32460) were used at 1:1000 dilution. The immunoreactive bands were detected using Chemi-Lumi One L or Chemi-Lumi One Ultra (Nacalai Tesque, Kyoto, Japan).

For mouse samples, 1–10 µg of protein per well was run on a 10% SDS gel. After transfer and blocking with 5% milk in TBST, immunoblotting was performed using the following primary antibodies: MSI1 (rat, 1:200, eBioscience #14H1), alpha-tubulin (rabbit, 1:1000, Cell Signaling #11H10). IRDye® 800CW goat anti-rat IgG (1:5000), IRDye® 680RD goat anti-rabbit (1:10,000) and IRDye® 800CW goat anti-mouse IgG (1:5000) were employed as the secondary antibodies (LICOR). All antibodies were diluted in 5% milk in TBST.
For human samples, denatured total cell protein (10 μg) was separated using 10% Bis-Tris gel electrophoresis and transferred to nitrocellulose membranes. Western blots were probed with the following antibodies: β-tubulin (rabbit; 1:50,000; Abcam #ab6046), GAPDH (mouse; 1:2000; Abcam #ab8245), MSI1 (rabbit; 1:2000; Abcam #ab52865) HIPK1 (1:500; Abcam #ab90103) (Table 1), Horseradish peroxidase conjugated with goat anti-rabbit IgG (1:20,000) or Licor anti-mouse (800 channel)/rabbit (700 channel) IgG were employed as the secondary antibodies (Bio-Rad). The bands were visualized with ImageStudio (Licor) or Chemidoc^TM MP Imaging Systems (Bio-Rad) using the ImageLab version 15.2.1 software.