Mouse anti beta actin antibody

We determined the expression levels of Cx43 and connective tissue growth factor (CTGF) protein in the bladder. Bladder extracts were prepared from a whole bladder homogenized in CelLytic^TM MT Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO, USA). Total protein concentration was quantified using a BCA Protein Assay Kit (Takara-Bio, Shiga, Japan) and a microplate reader. Thirty μg of homogenate protein was loaded and separated by 10% SDS-PAGE gel and transferred onto Immobilon-P transfer membranes (Merck Millipore Japan, Tokyo, Japan). The membranes were blocked for 2 h with 3% skim milk in Tris-buffered saline-Tween20 (TBS-T) at 4 ℃, and sequentially incubated with rabbit anti-Cx43 antibody (1:500, Novus Biologicals), rabbit anti-CTGF antibody (1:1000, GeneTex) or mouse anti-beta-actin antibody (1:20000, Proteintech) at 4 ℃ overnight. After washing with TBS-T, membranes were incubated for 1 hour with HRP-conjugated anti-rabbit-IgG (1:2000) or anti-mouse-IgG (1:4000) at room temperature. After washing membranes with TBS-T, protein bands were visualized with ImmunoStar^® Zeta (Wako). The expression levels of Cx43 and CTGF proteins per beta-actin protein of the Sham group were regarded as 1.0.

Mouse anti-E-cadherin and anti-vimentin antibodies were purchased from BD Technologies (La Jolla, CA, USA). Rabbit anti-ZO1 antibody was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-Snail antibodies, DMOG and LY294002 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against Slug, GSK3β, p-GSK3β (Ser9), Akt, p-Akt (Ser473), STAT3, and p-STAT3 (Thr705) were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-HIF-1α antibody was purchased from Abcam (Cambridge, MA, USA), and mouse anti-beta-actin antibody was obtained from Proteintech Group (Chicago, IL, USA). Goat anti-mouse (W4021) and goat anti-rabbit (W4011) peroxidase-conjugated secondary antibodies were obtained from Pierce Biotechnology (Rockford, IL, USA). The following reagents were used: anti-IL-11 neutralizing antibody (LifeSpan Technology, Newton, MA, USA), recombinant human IL-11 (rhIL-11) (Prospecbio, Rehovot, Israel), and Akt inhibitor V (Millipore, Billerica, MA, USA). All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich (St. Louis, MO, USA).