Ab57169

Protein lysates were quantified and separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After the separation, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc.), and immunoblotted with a primary antibody, followed by an incubation with a secondary antibody. The luminescence was visualized on the Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd.). The following antibodies were used: AZIN1 (1:1000, ab57169; Abcam), β-tubulin (1:5000, AP0064; Bioworld), goat anti-mouse IgG (1:5000, HS201; TransGen Biotech), and goat anti-rabbit IgG (1:5000, HS101; TransGen Biotech).

The cells were fixed in 100% methanol (10 min), permeated with 0.5% Triton X-100/PBS for 10 min, and blocked with 10% BSA/0.2% PBS-Tween for 30 min at 37°C. The cells were then incubated with the AZIN1 antibody (ab57169; Abcam) used at a 1/100 dilution overnight at 4°C. The secondary antibody (pseudo-colored red) was Alexa Fluor ® 594 goat anti-mouse IgG (Life Technologies Corporation), which was used at a 1/1000 dilution for 45 min at room temperature. ProLong ® Gold Antifade Reagent with DAPI (Life Technologies, USA) was used to stain the cell nuclei (pseudo-colored blue).