Hrp conjugated anti rabbit igg

RAW264.7 cells (6 × 10⁵/well) were seeded into 24-well plates and treated with DMEM media only or 5 μg/mL of LPS or with indicated concentrations of compounds for 24 h. Cells were lysed in sodium dodecyl sulfate (SDS) sample buffer containing protease inhibitor cocktail (Roche Life Science, Mannheim, Germany). The resulted proteins were resolved by SDS-polyacrylamide gel electrophoresis and then subjected to the following antibodies: anti-mouse IKK-α, anti-mouse phospho-IKK-α, anti-mouse IκB-α, anti-mouse NF-κB p65, anti-mouse phospho-NF-κB p65 antibodies (Cell Signaling Technology, Beverly, MA), and HRP-conjugated mouse anti-GAPDH (Kangcheng, Shanghai, China). Signals were detected with an HRP-conjugated anti-rabbit IgG (Bio-Rad, Richmond, CA, USA) using an ECL system (Amersham Biosciences, Buckinghamshire, UK). The uncropped and unprocessed scans of indicated blots were supplied as Supplementary Figure 99 in the Supplementary Information.

Total protein extracts were prepared with sodium dodecyl sulfate buffer and uniformed by the Pierce BCA protein assay kit (Thermo Fisher Scientific). Equal protein amounts were subjected to 10% SDS–PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with SuperBlock™ T20 (PBS) blocking buffer (Thermo Fisher Scientific) and then incubated overnight at 4 °C with rabbit, mouse or rat primary antibodies (Supplementary Table S2). The signals were analyzed with HRP-conjugated anti-rabbit IgG (Bio-Rad), HRP-conjugated anti-mouse IgG (Kangchen, Shanghai, Chinia), or HRP-conjugated anti-rat IgG (Cell Signaling Technology) and further visualized by SuperSignal^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) under ChemiDoc™ MP Imaging System (Bio-Rad).

CLPr was prepared from cacao liquor and its composition was previously described.^{(17 (link),19 (link))} Glucose was measured using a commercially available kit [Labassay Glucose Wako kit (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan)]. Antibodies against β-actin, AMPKα, phospho-AMPKα (thr172), phospho-CaMKK2 (ser511), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), liver kinase B1 (LKB1) and phospho-LKB1 (ser428) were purchased from Cell Signaling Technology Co. (Denver, MA). Antibodies against CaMKK2 and GLUT4 were purchased from Abcam (Hercules, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG, HRP-conjugated anti-goat IgG antibodies, anti-insulin receptor (IR), and anti-Lamin B were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated anti-rabbit IgG was from Bio-Rad Laboratories Inc. (Hercules, CA). All other reagents used were of the highest grade available from commercial sources.

Western blots were used to quantify the amount of tyrosine hydroxylase (TH), dopamine transporter (DAT), human α-synuclein, and actin present in samples of striatal tissue and were performed as described previously (Caudle et al., 2006 (link)). Membranes were incubated overnight in a TH monoclonal antibody (1:1000; Chemicon) and detected using a goat anti-rabbit horseradish peroxidase secondary antibody (1:5000) and visualized by enhanced chemiluminescence using an ECL kit (GE). Membranes were stripped for 15 min at room temperature with Restore Stripping Buffer (Thermo Fisher, Rockford, IL, USA) and sequentially re-probed with DAT (1:5000; Chemicon), and human α-synuclein (1:500; Abcam) antibody. Actin blots were used to ensure equal protein loading across samples.
For MPTP sample analysis, striata were mechanically homogenized in RIPA buffer and cytosolic fractions taken prepared for reducing, denaturing SDS-PAGE. Proteins were separated on 10.5–14.5 % gradient gels and transferred using a Trans-Blot Turbo transfer system (Bio-Rad, UK). Membranes were blocked and probed with polyclonal rabbit anti-tyrosine hydroxylase (Millipore, AB152) or polyclonal rabbit anti β-actin antibody (Abcam, AB8227) and visualized with HRP conjugated anti-rabbit IgG (Bio-Rad, UK). Densitometry analysis was performed using ImageJ software.