Mouse monoclonal α tubulin antibody

Following incubations, cells were lysed in RIPA buffer (Boston Bioproducts, Ashland, MA) with PhosSTOP and cOmplete Protease Inhibitor cocktail (Roche Life Science, Indianapolis, IN). Cell lysates were resolved by AnyKDa SDS PAGE gel (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad, Hercules CA). Blots were blocked for 1 h with 5% BSA in TBST. Membranes were incubated with primary antibody (1:1000 – 1:3000) in TBST overnight at 4°C and probed with the respective secondary antibody (1:2000) (Cell Signaling Technology, Danvers, MA) for 1 hr at room temperature. Membranes were then incubated with WesternSure ECL (Li-COR Biosciences, Lincoln, NE) or Pierce ECL (Thermo Fisher Scientific, Cambridge MA) and imaged using a Bio-Rad ChemiDoc XRS (Bio-Rad, Hercules, CA). Antibodies used in this study included rabbit monoclonal NF-κB phospho p-65 (Cell Signaling Technology, Danvers, MA), rabbit monoclonal SIGIRR (GeneTex, Irvine, CA), rabbit polyclonal LL-37 (Abcam, Cambridge, MA), rabbit polyclonal vinculin (Cell Signaling Technology, Danvers, MA) and mouse monoclonal α tubulin antibodies (Sigma Aldrich, St Louis, MO). Densitometry was performed using ImageJ software (NIH, Bethesda, MD) and normalized to unstimulated control.

Total protein extracts from the cells were prepared, as previously described (Santra et al. 2011 (link)). The protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis for Western blot analysis. For Western blot analysis, rabbit antiserum for MBP (1:200; Dako, Carpinteria, CA, USA), monoclonal antibodies (1:500) for EGFR and phosphorylated EGFR; monoclonal antibodies (1:1000) for phosphorylated ERK1, c-JUN and FOG-2 (Santa Cruz Biotechnology, Dallas, Texas, USA); rabbit polyclonal antibodies (1:500) for Grb2, Mig-6, Pten, p53 (Cell Signaling Technology, Danver, MA, USA) and mouse monoclonal α-tubulin antibodies (1:5000; Sigma, St Louis, Mo) were used. Donkey anti-goat, anti-rabbit, and anti-mouse horseradish peroxidase (1: 5000; Jackson ImmunoResearch Labs, West Grove, PA, USA) were used as secondary antibodies. Each experiment was repeated at least five times. The protein bands were quantified based on histogram analysis relative to gel loading marker α-tubulin at least 5 independent experiments.