α tubulin antibody

After the corresponding experimental treatment, 3D4/21 cells were fixed with 4% paraformaldehyde at room temperature for 30 min, treated with 1% Triton X-100 for 10 min, and blocked with bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 1 h. The samples were then incubated overnight with primary antibody (α-Tubulin antibody, abcam Co., Ltd., Cambridge, MA, USA) at 4 °C. The cells were gently washed with PBST and incubated for 1 h, in the dark, with fluorescently labeled secondary antibodies (abcam Co., Ltd., Cambridge, MA, USA). After gently washing the cells 3 times with PBST, DAPI was added to counterstain cell nuclei. Samples were observed and photographed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

TSCs were grown on coverslips in 6-well plates at a density of 0.05 million for undifferentiated control cells and 0.5 million for differentiation into TGCs or for treatment with Aurora A inhibitor. Cells on coverslips were washed with PBS and fixed with 4% formaldehyde for 15 min at room temperature. The cells were then washed again with PBS, followed by permeabilization and blocking with 0.1% Triton X solution prepared in 2% BSA for 20 min at room temperature. After permeabilization, cells were washed with PBS and incubated with phalloidin Texas Red (Invitrogen) or phalloidin Alexa Fluor 488 (Thermofisher Scientific) or α-tubulin antibody (Abcam, 1:500 dilution) for an hour. For tubulin staining, cells were washed and incubated for another hour with AlexaFluor 488-labeled secondary antibody. The coverslips for both phalloidin and tubulin staining were washed five times with PBS, stained with DAPI (in the penultimate wash), and mounted on the slides using Fluoromount™. Images were taken using a confocal microscope (Nikon Model C2+).

CEK cells were infected with identical doses (200 μL of PBS containing 10⁸ copies) of rYN or rYN-Δ5a. At 12, 24, 36, or 48 hpi, the cells were washed with cold PBS and harvested in 80 μL of 2× lysis buffer. After being incubated on ice for 30 min, the cells were sonicated for 1 min and then boiled. Equal protein amounts were separated by SDS-PAGE, and proteins were detected via Western blotting using specific antibodies against IBV N (Hytest, Finland), β-actin (Merck Millipore, Burlington, MA, USA), α-tubulin antibody (Abcam, Cambridge, UK), and histone H3 antibody (Abcam). Images were taken with the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Protein signal intensities were normalized and quantified using ImageJ software (NIH, Bethesda, MD, USA).

CEK cells were infected with identical doses (200 μL of PBS containing 10⁸ copies) of rYN or rYN-Δ5a. At 12, 24, 36, or 48 hpi, the cells were washed with cold PBS and harvested in 80 μL of 2× lysis buffer. After being incubated on ice for 30 min, the cells were sonicated for 1 min and then boiled. Equal protein amounts were separated by SDS-PAGE, and proteins were detected via Western blotting using specific antibodies against IBV N (Hytest, Finland), β-actin (Merck Millipore, Burlington, MA, USA), α-tubulin antibody (Abcam, Cambridge, UK), and histone H3 antibody (Abcam). Images were taken with the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Protein signal intensities were normalized and quantified using ImageJ software (NIH, Bethesda, MD, USA).

Immunofluorescence was performed as described [28 (link)]. The primary antibody was anti-α-Tubulin (Cell Signaling; mouse monoclonal; 1:2000) and the secondary antibody was Alexa488-conjugated anti-mouse IgG (Molecular Probes; 1:500). The slides were mounted in VectaShield fluorescence mounting medium containing 4, 6-diamidino-2-phenylindole (Vector Laboratories). Images were analyzed using a Zeiss confocal laser scanning microscope. Alternatively, cells were stained with α-tubulin antibody (1:50, mouse monoclonal; Abcam) followed by a CY5-conjugated secondary anti-mouse antibody (1:200; Abcam). The cover slips were mounted with Invitrogen Anti Fade Mounting Medium containing DAPI and imaged using a Zeiss AxioImager.Z1 microscope at 63x magnification.