Mini protean tgx precast gel

Protein extracts were prepared from human primary motor cortex tissue samples (control, n = 8, PD, n = 9), provided by Banner Sun Health Research Institute Brain and Body Donation program [2 (link)]. The protein concentration of each sample was determined using bicinchoninic acid assay (BCA, Pierce Biotechnology, Rockford, IL) according to the manufacturer directions. Proteins (30 μg per well) were separated on 4-20% Mini-PROTEAN TGX pre-cast gels (Bio-Rad Laboratories, Los Angeles, CA) and transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were probed with rabbit anti-histone H3 (1:1,000, Cell Signaling, Danvers, MA), rabbit anti-acetyl-histone H3 (1:20,000, EMD Millipore), rabbit anti-acetyl-histone H3K9 (1:500, EMD Millipore), goat anti-rabbit acetyl-histone H3K14 (1:1,000, EMD Millipore), goat anti-rabbit acetyl-histone H3K18 (1:25,000, EMD Millipore), or goat anti-rabbit acetyl-histone H3K23 (1:200,000, EMD Millipore). The secondary antibody was goat anti-rabbit IR Dye 800CW (1:15,000, LI-COR Biosciences, Lincoln, NE). Immunoblots were visualized with Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Bands of interest were analyzed and quantified using Scion Image. A researcher blind to the treatment groups conducted the Western blots, blot visualization, and quantification of bands of interest. All assays were performed in triplicate.

Fractionated samples were treated with 4× SDS polyacrylamide gel electrophoresis (SDS-PAGE) Sample Buffer (Bio-Rad) containing 5 mM Aldrithiol and boiled at 95°C for 5 min. Next, β-mercaptoethanol was added to 2.5% concentration, and after re-boiling samples were subjected to SDS-PAGE using 4%–20% Mini-Protean TGX Precast Gels (Bio-Rad) in TRIS/glycine/SDS buffer (Laemmli, 1970 (link)). Proteins were transferred onto a nitrocellulose membrane in 25 mM TRIS, 192 mM glycine, and 20% methanol at 100 V for 60 min. The amount of protein was estimated by standard Ponceau S staining. Membranes were blocked with 2% bovine serum albumin (BSA) in TTBS (TBS + 0.1% Tween-20) at room temperature for 1 h and further labeled with specific antibodies, namely, anti-GP1 (anti-Rgp), anti-Kgp, anti-RgpB, anti-PPAD, anti-CTD, and anti-RagB diluted in blocking solution. Signals were detected using Pierce ECL Western Blotting Substrate (Thermo) and visualized on X-Ray film (AGFA).

The APC/C holoenzyme from G1 synchronized CSCs and NSTCs was immunoprecipitated as described above with resulting immmunoprecitated proteins taken on beads for processing by the Proteomics Shared Resource at The Ohio State University Comprehensive Cancer Center for protein identification and quantification of protein abundance. For post-translational modification analysis of CDH1, extracts were prepared for G1 synchronized CSCs and NSTCs by resuspending cell pellets in extract buffer (20 mM Tris-HCl, pH 7.2, 2 mM DTT, 0.25 mM EDTA, 5 mM KCl, 5 mM MgCl₂) followed by two rounds of freeze-thaw and passage through a needle. Extracts were supplemented with ATP and an energy regenerating system. 5 ug of recombinant GST-CDH1 fusion protein was then incubated with the extracts for 1 hour at 30°C and then captured on Glutathione beads. Samples were resolved on 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad Laboratories, Inc.) and visualized with GelCode Blue Stain Reagent as per manufacturer instructions (ThermoFisher Scientific). The GST-CDH1 band was then cut from the gel and processed by the Lerner Research Institute Mass Spectrometry Laboratory for Protein Sequencing for proteomic analysis and quantification of post-translational modifications on GST-CDH1.