Hrp conjugated goat anti rabbit and goat anti mouse antibodies

The rabbit polyclonal antibody specific to ADD1 pT724 was generated using synthesized phospho-peptides KLH-CKKKKFRTpPSFLKKS (pT724 peptide) as the antigen (AllBio, Inc., Taichung, Taiwan). The rabbit polyclonal anti-ADD1 pS726 (sc-16736), anti-ADD1 (sc-25731), anti-PKCδ (sc-937), and anti-cortactin (sc-11408) antibodies and mouse monoclonal anti-Cdk5 (sc-249) and anti-β-tubulin (sc-5274) antibodies were purchased from Santa Cruz Biotechnology. The rabbit polyclonal anti-FLAG (F7425) and anti-Cdk5 pY15 (SAB4504276) antibodies and mouse anti-β-actin (A5441) antibody were purchased from Sigma-Aldrich. The rabbit monoclonal anti-p35 (C64B10) antibody and rabbit polyclonal anti-PKCδ pY311 were purchased from Cell Signaling Technology. The mouse monoclonal anti-HA (MMS-101P) antibody was purchased from Covance. The HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. Dulbecco’s modified Eagle’s medium (DMEM), Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies, and Lipofectamine were purchased from Invitrogen Life Technologies. Purified Cdk5/p35 was purchased from EMD Millipore. EGF was purchased from R&D Systems. G418 and puromycin were purchased from Merck. Matrigel was purchased from BD Transduction Laboratories. The muscle actin was purchased from Cytoskeleton, Inc.

To prepare protein lysates, cells were washed once with ice-cold PBS and lysed in 1X Cell Extraction Buffer (Invitrogen) supplemented with protease inhibitor tablet (Roche Diagnostics, Indianapolis IN) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Protein concentration was determined using BCA Protein Assay (Thermo Fisher Scientific, Waltham MA). Equal amounts of proteins were resolved by 10% Bis-Tris gels in 1X MOPS running buffer (Invitrogen) and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham MA). Antibodies directed against the following proteins were used: p-ERK, ERK1/2, p-MEK, MEK, cyclinD1, p27, cleaved PARP (Cell Signaling, Danvers MA), p-p90RSK (Abcam, Cambridge MA), GAPDH (Millipore-Sigma, Billerica MA), p90RSK (Thermo Fisher Scientific, Waltham MA). Antigen-antibody interaction was detected with HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies (Jackson ImmunoResearch, Westgrove, PA) using enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, Waltham MA) or with IRDye 800 conjugated, affinity purified anti rabbit IgG (LI-COR Biosciences, Lincoln, NE) and Alexa Fluor 680 goat anti mouse IgG (Thermo Fisher Scientific, Waltham MA) secondary antibodies using a Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).