Anti mouse antibody against β tubulin

Protein extracts from Vero cells were separated by electrophoresis in 12% acrylamide–bisacrylamide gels, or 7% to detect PIKfyve. Separated proteins were transferred to nitrocellulose membranes, and proteins were detected with specific antibodies in each case. As a secondary antibody, anti-mouse IgG (GE Healthcare, Little Chalfont, UK) or anti-rabbit IgG (Bio-Rad, Oslo, Norway) conjugated to horseradish peroxidase, were used at a 1:5000 dilution. Precision Protein StrepTactin-HRP Conjugate (Bio-Rad) was used to reveal the ladder Precision Plus Protein WesternC (Bio-Rad). As a load control in WB analysis, an anti-mouse antibody against β-tubulin (Sigma) 1:2000 was used. Finally, bands obtained after development with ECL reagent were detected on the Molecular Imager Chemidoc XRSplus Imaging System (Bio-Rad, Hercules, CA, USA). Bands were quantified by densitometry, and data were normalized to control values using Image system software.

Cells were harvested in Laemmli sample buffer (Life technologies), heated for 5 min at 95 °C and resolved by SDS-PAGE in 12% polyacrylamide-bisacrylamide gels. Afterwards, separated proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and the non-specific antibody binding sites were blocked with skimmed milk diluted in PBS and then incubated with the specific primary and HRP (Horseradish peroxidase)-conjugated secondary antibodies. Antibodies used for western blotting included: anti-rabbit polyclonal against BAG3 1:500 (Proteintech). As a loading control an anti-mouse antibody against β-tubulin (Sigma) 1:2000 was used. As secondary antibodies, anti-mouse IgG (GE Healthcare, Piscataway, NJ, USA) or anti-rabbit IgG (Bio-Rad) conjugated to horseradish peroxidase were used at a 1:5000 dilution. Precision Protein StrepTactin-HRP Conjugate (Bio-Rad) was used to reveal the ladder Precision Plus Protein WesternC (Bio-Rad). Finally, bands obtained after development with ECL reagent (GE Healthcare, Piscataway, NJ, USA) were detected using a Chemidoc XRSplus Imaging System (Bio-Rad). Band densitometry was performed with Image Lab software (Bio-Rad Inc: 2011) and data were normalized to loading control values.