Mouse antihuman tubb1 antibody

Whole protein lysate was isolated using RIPA buffer [150 mMNaCl, 50 mMTris-HCl (pH = 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] completed with 100X protease/phosphatase inhibitor cocktail (Thermo Fisher, Massachusetts, USA), and protein concentration was measured by BCA protein assay kit (Thermo Fisher). Each lysate from different samples was separated by using Mini-PROTEAN TGX 10% precast gel (Bio-Rad, California, USA) and transferred to nitrocellulose membrane using Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA (Sigma–Aldrich) in TBS-T and then incubated with a mouse antihuman TUBB1 antibody (OriGene, TA506654; 1:3000 dilution) at 4 °C overnight, an anti-Flag HRP antibody (Abcam, ab49763; 1:1000 dilution, Cambridge, UK) at room temperature for 1 h, a rabbit antihuman GAPDH antibody (Cell Signaling, 5174; 3:10000 dilution) at room temperature for 1 h, or a rabbit antihuman β-actin antibody (Cell Signaling, 8457; 1:1000 dilution) at room temperature for 1 h. Upon washing with TBS-T, membranes were incubated with compatible HRP-conjugated antimouse IgG (Cell Signaling, 7076; 1:5000 dilution) or antirabbit IgG (Cell Signaling, 7074; 1:5000 dilution) secondary antibodies at room temperature for 1 h. Protein bands were detected using ECL Plus Western blotting substrate (Thermo Fisher) and ChemiDoc XRS+ imaging system (Bio-Rad).

For immunofluorescence (IF) staining, peripheral blood smears or the transfected HeLa cells that were seeded on the chamber slides were fixed with absolute methanol at –20 °C for 15 min. After washing with PBS-T (0.1% Triton-X-100), slides were incubated in blocking/permeabilization solution (2% skim milk in PBS-T) at room temperature for 1 h. Transfected HeLa cells were incubated with mouse antihuman α-TUBULIN antibody (Santa Cruz, sc-5286; 1:100 dilution, Texas, USA) and rabbit antihuman Flag antibody (Sigma–Aldrich, F7425; 1:100 dilution). Slides of fixed blood smears were incubated with mouse antihuman TUBB1 antibody (OriGene, TA506654, 1:150 dilution, Maryland, USA) and rabbit antihuman α-TUBULIN antibody (Cell Signaling, 2125; 1:100 dilution, Massachusetts, USA). After washing, slides were incubated with corresponding anti-IgG antibodies with Alexa-488 (Cell Signaling, 4412; 1:500 dilution) or Alexa-555 fluorophore (Cell Signaling, 4409; 1:500 dilution) at room temperature for 1 h. Slides were mounted with DAPI solution (1 µg/10 mL prepared in ddH2O) for nuclei staining. Stained cells were visualized under LSM 880 laser scanning confocal microscope (Zeiss, Göttingen, Germany).