Rabbit monoclonal anti actin

Cells grown in the presence or absence of IAA at 30 °C for 4 h were harvested. Whole protein extracts were prepared and protein concentrations were quantified by the Bradford assay48 (link). Western blot analysis was performed as described34 (link). Primary antibodies used were mouse monoclonal anti-α-tubulin (Sigma) diluted at 1:500, mouse monoclonal anti-β-tubulin TU27B kindly provided by B. R. Oakley (The Ohio State University, Columbus, OH) diluted at 1:200, and rabbit monoclonal anti-actin (Sigma) diluted at 1:100. Secondary antibodies used were goat anti-mouse IgG (H+L), HRP conjugate (Promega) diluted at 1:1,000, horseradish peroxidase-conjugated goat anti-mouse IgG (DAKO, Glostrup, Denmark) diluted at 1:1,500 and horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (Bio-Rad) diluted at 1:3,000 for anti-α-tubulin, anti-β-tubulin, and anti-actin, respectively.

Cells were homogenized in RIPA plus buffer [Phosphate Buffered Saline (PBS); NP-40 1%; Na deoxycolate 0.5%; SDS 0.1%; EDTA 1 mM; NaF 50 mM; NaVO₃ 5 mM] containing a cocktail of EDTA-free protease inhibitors (Roche). Protein concentration was determined by the Bradford method by using the BCA Protein Assay Kit (Pierce) and bovine serum albumin as a standard. Twenty micrograms of protein were loaded and subjected to electrophoresis in 10% SDS-PAGE gels (Invitrogen) and transferred onto PVDF membranes (Bio Rad). After 1 h of blocking (LICOR Biosciences) membranes were incubated overnight at 4°C with a rabbit polyclonal anti-PKM2 primary antibody (Cell signaling; 1:1000) or with a mouse monoclonal anti-p53 (Abcam; 1:500). Rabbit monoclonal anti-Actin (1:2000) and mouse monoclonal anti-α-Tubulin antibodies (1:15000) (both from Sigma Aldrich) were used as internal controls. Membranes were incubated with IRDye rabbit and mouse secondary antibodies (1:15000) (LICOR Biosciences) for 45 minutes protected from light. Membranes were scanned by using Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences).