Anti β tubulin antibody

Reagents sources were as follows: recombinant mouse canstatin (produced by Escherichia coli as described previously)^{14 (link)} and TGF-β1 (PeproTech, Rocky Hill, NJ, U.S.A).
Antibody sources were as follows: anti-NFATc4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), anti-type I collagen antibody (Rockland Immunochemicals, Gilbertsville, PA, U.S.A), anti-α-SMA antibody (Dako, Glostrup, Denmark), anti-β-tubulin antibody (Wako, Osaka, Japan), anti-rabbit IgG horseradish peroxidase linked whole antibody and anti-mouse IgG horseradish peroxidase linked whole antibody (Cell signaling Technology, Danvers, MA, U.S.A.).

Cultured tissue cells were lysed in lysis buffer supplemented with a protease inhibitor cocktail and disrupted on ice using a sonicator. The protein concentration of cell lysates was assessed using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as a standard. Each cell lysate (20 µg) was subjected to SDS-PAGE using a 5-20% gel (Wako, Osaka, Japan) and transferred to a PVDF membrane. After blocking with PVDF-blocking reagent (Toyobo, Osaka, Japan), 1 μg/mL of purified polyclonal antibody for each antigen in Can Get Signal 1 (Toyobo) were incubated with the membrane. Immunoreactive antigens were detected using anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Tokyo, Japan) and Western Lightning Plus ECL (PerkinElmer, Waltham, MA, USA). The control monoclonal antibodies to MAGE-A4 (clone: E701U, Cell Signaling Technology, Tokyo, Japan) and XAGE-1b (clone: USO9-13) (47 (link)) were employed for the validation of the specificity of polyclonal antibodies. The membrane was reprobed with an anti-β-tubulin antibody (Wako).