Acetylated α tubulin clone 6 11b 1

A rabbit polyclonal antibody against DDDDK-tag was used as the anti-Flag antibody (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan). Mouse monoclonal antibodies against α-tubulin (DM1A (epitope; 426–450 residues), NeoMarkers, Fremont, CA, USA), Cy3-conjugated-β-tubulin (both antibodies used for general tubulin staining), and acetylated-α-tubulin (clone 6-11B-1) (Sigma, St. Louis, MO, USA), were used. Human anti-centromere antibody (Antibodies Inc., Davis, CA, USA) was also used. DAPI (4,6-diamidino-2-phenylindole dihydrochloride hydrate) (Sigma), and tubacin (Enzo Life Sciences, Farmingdale, NY, USA) were used. Salicylate (sodium salicylate) was purchased from Sigma. NAP peptide (Biorbit, San Francisco, CA, USA) was aliquoted and stocked as a 1 mM solution in 75% dimethyl sulfoxide at −20 °C.

Immunospecificity of the mAbs raised against chicken α-tubulin (clone N356; Amersham Pharmacia Biotech, Piscataway, NJ. USA), sea urchin (S. purpuratus) acetylated α-tubulin (clone 6-11B-1, Sigma, St. Louis, MO. USA), and sea urchin (S. purpuratus) β-tubulin (clone 2-28-33, Sigma) were diluted 1:1000 in Tris-buffered saline with 1 % Tween-20 (v/v) (TBST). Rabbit anti-Sp-Syn pAb and anti-Hp-ECPN pAbs were diluted 1:1000 and 1:750 respectively in TBST. Rabbit pre-immune serum and mouse pre-immune serum (diluted 1:1000 in TBST) were applied as negative immunoblotting controls in lyophilized 4-arm and 6-arm plutei. The samples were diluted in 0.1 M sample buffer with 2-mercaptoethanol and separated by 10 % uniform sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, blotted to a polyvinylidene difluoride membrane, blocked with 5 % (w/v) skim milk diluted in TBST, and incubated with the above described pAbs or mAbs for 2 h at ambient temperature (AT). The primary antibodies were detected by incubation with alkaline phosphatase (AP)-tagged anti-mouse or rabbit IgG pAbs (Sigma) diluted 1:15,000 in TBST for 2 h at AT and visualized with the chromogenic reagents nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate (Promega, Madison, WI, USA) diluted in AP buffer (pH 9.5) according to the manufacturer’s instructions.