Anti mouse hrp linked antibody

Mouse monoclonal anti-β- and anti-γ-actin antibodies directed against epitopes present on the N-terminus of β- or γ-actin were purchased from Sigma-Aldrich (clone AC-15 or clone 2–2.1.14.17, respectively) or from AbD Serotec (clone 4C2 or clone 2A3 respectively). Mouse monoclonal anti-GFP antibodies were obtained from Santa Cruz Biotechnology. Phalloidin conjugated with Alexa Fluor 568 and DNase I conjugated with Alexa Fluor 594 (detecting unpolymerized actin) were purchased from Invitrogen. Rabbit anti-myosin II antibodies as well as mouse monoclonal antibodies against ezrin were purchased from Sigma-Aldrich. Secondary anti-rabbit or anti-mouse antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 568 were obtained from Invitrogen, while anti-mouse HRP-linked antibodies were from Cell Signaling. Fetal bovine serum (FBS), glutamine, penicillin/streptomycin, trypsin, alpha-MEM media and Lipofectamine 2000 were obtained from Invitrogen. DNase I from bovine pancreas and DNA from calf thymus were purchased from Sigma-Aldrich. Collagen type I was purchased from Corning, while epidermal growth factor (EGF) was obtained from BD Biosciences. Dako fluorescent mounting medium was obtained from Dako. All other reagents were classified as analytical grade reagents.

Protein samples in appropriate buffers according to the experiments were diluted in Laemmli buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 0.05% bromophenol blue, pH 6.8) and heated 10 min at 100 °C. After electrophoresis in a 10% acrylamide SDS-PAGE gel, proteins were transferred onto a nitrocellulose membrane at 100 V for 1 h in a Tris/glycine buffer supplemented with 10% ethanol. After blocking of the nitrocellulose membrane with 0.05% TBS Tween-20 (TBS-T) supplemented with 5% milk for 1 h at room temperature, membrane was probed with primary antibodies diluted at 1:200 for NEU-1 (NEU-1 F8 Santa Cruz) and at 1:750 for β actin (Santa Cruz Biotechnology, Heidelberg, Germany) in TBS-T with 3% BSA overnight at 4 °C. Membrane was then washed in TBS-T and incubated with HRP-linked secondary antibodies diluted at 1/10,000 in TBS-T with 5% milk at room temperature. Anti-mouse HRP-linked antibodies (Cell Signaling, Danvers, MA, USA) were used for protein detections. Chemiluminescent protein detection was done using ECL Prime and ODYSSEY Fc (LI-COR Biosciences-GmbH, Bad Homburg, Germany) hardware and the Image Studio software.

Preparation of whole cell lysates and nuclear extracts as well as electrophoresis were performed as described [75 (link),76 (link)]. The antibodies listed in Table 1 were used according to the manufacturer’s instructions. Primary antibodies were incubated overnight at 4 °C and detected by anti-mouse HRP-linked antibodies (Cell Signalling, Frankfurt, Germany) at room temperature (RT) for 1 h. After washing in TBST, blots were developed with SuperSignal West Dura Extended Duration Substrate (Perbio Sciences, Bonn, Germany).