Goat anti mouse hrp

Protein samples from the GST-pulldown, immunoprecipitation assays, and flux assays were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies overnight at 4 °C. After three washes, membranes were incubated with secondary antibodies and conjugated with horseradish peroxidase for an hour. Signals were visualized with ECL using Advansta WesternBright ECL (K-12045-D50). The antibodies using in the experiment were used: Flag (Sigma, F1804, 1:10000), GFP (Santa Cruz Biotechnology, sc-9996, 1:10000), LC3B (Cell Signaling Technology, #2775, 1:1000), GABARAP (Cell Signaling Technology, #13733, 1:1000), donkey anti-rabbit HRP (Santa Cruz Biotechnology, sc-2313, 1:10000) and goat anti-mouse HRP (Santa Cruz Biotechnology, sc-2005, 1:10000). In order to quantify the intensity of the western blot band, the area of each band was quantified using the ImageJ program. In the same manner, the Band Quantitation was obtained from three independent experiments. All statistical data were calculated and graphed using GraphPad Prism5 (GraphPad, Inc., La Jolla, CA, USA).

The protein concentration in cell lysate was assessed using BCA Protein Assay Reagent (Thermo Fisher Scientific, Rockford, IL, USA). Authors used the respective primary antibodies (p53: mouse monoclonal antibody, Cat# SC-99, Santa Cruz Biotechnology; PARP: mouse monoclonal antibody, Cat# SCBSC-8007; Cytochrome c: mouse monoclonal antibody, Cat# SCBSC-13156; Bax: mouse monoclonal antibody, Cat# 336400, Life Technologies; and GAPDH: mouse monoclonal antibody, Cat# MA5-15738, Invitrogen) and the horseradish peroxidase-conjugated secondary antibody (Goat anti-mouse HRP, Cat# SC-2005, Santa Cruz Biotechnology) for the western blot experiments and followed the protocol explained by George et al. [18 (link)].

Bone marrow cells from p38 knockout or control animals were lysed in 100 µL complete lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 100 mM NaF, 1% Triton X-100, 100 mM dithiothreitol, 100 mM Na₃VO₄ and 1× Protease Inhibitor (complete Mini, Roche, Basel, Switzerland)) for 20 min on ice. The protein concentration in the supernatant was determined using the Bradford assay (Protein Assay Dye Reagent Concentrate, Bio-Rad, Hercules, CA, USA). Protein samples were separated on a 12% polyacrylamide gel and blotted onto a nitrocellulose membrane (Whatman Protran BA 85 Nitrocellulose). The membrane was blocked in 5% milk in TBST for 1 h at room temperature. Primary antibody staining (p38, clone: E229, Abcam, Cambridge, UK; Cbl, clone: A-9, Santa Cruz, Dallas, TX, USA and Lnk, clone: A-12, Santa Cruz, Dallas, TX, USA) was performed overnight at 4 °C. A horseradish peroxidase-conjugated secondary antibody (goat-anti-rabbit-HRP or goat-anti-mouse-HRP, Santa Cruz, Dallas, TX, USA) was applied for 1 h as a secondary antibody. GAPDH-HRP (clone: GT239, Genetex, Irvine, CA, USA) was used as loading control. Signals were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and the FusionX detection system (Peqlab, Erlangen, Germany).

Olfactory epithelia were dissected from E17.5 embryos and protein lysates prepared in Universal Lysis Buffer. Electrophoresis was performed using 8% Tris-Chloride gels and proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), and detected using antibodies to total RB (BD-Pharmingen, San Diego, CA, 1:250) and GAPDH (Santa Cruz, 1:1000). The secondary antibodies used are: goat anti-mouse HRP (Santa Cruz 1:2500) and goat anti-rabbit HRP (Santa Cruz, 1:2500).