Western Blot Cell-Based Dose Response to Identify Activators of Alpha-Synuclein Translation in H4 Cells
Jack T. Rogers, Massachusetts General Hospital, firstname.lastname@example.org, 617-726-8838, Charlestown, MA ..more
BioActive Compound: 1
Depositor Specified Assays
Broad Institute MLPCN Alpha-Synuclein 5'UTR Project
Project ID: 2023
Keywords: alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region
Primary Collaborators (and laboratory where this assay was performed):
Jack T. Rogers, Massachusetts General Hospital, email@example.com, 617-726-8838, Charlestown, MA
The goal of this project is to identify novel small molecule probes that increase alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a novel probe for alpha-synuclein translational expression. The 5'UTR of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Increased levels of alpha-synuclein have been directly linked to Parkinson's disease. Probes that can successfully increase alpha-synuclein expression levels as measured in this luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5'UTR stem-loop to confirm that probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein 5'UTR mRNA stem-loop and also in cells containing the amyloid precursor protein 5'UTR mRNA stem-loop, both of which are fused to a luciferase reporter.
Assay Overview: In the Western blot orthogonal screen, confirmation of alpha-synuclein translation activators was sought. The Western used H4 neuroblastoma cells that were not transfected. Compound was tested in 3-point dose in an effort to observe a dose-dependent effect.
Expected Outcome: Activators of alpha-synuclein translation should increase alpha-synuclein protein levels without affecting actin levels.
Cell Culture and Preparation of Lysates:
1. Human H4 neuroglioblastoma cells were cultured in Dulbeccos modified essential medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Bio-Whittaker, Walkerville, MD). Cells were exposed to increasing concentrations of compound (0, 0.1, 1, 10 micromolar) for 48 hours.
2. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT).
1. Western blotting for alpha-synuclein was performed using mouse monoclonal anti-alpha-synuclein (BD Transduction Laboratories), and anti-beta-actin (Chemicon).
2. The blots were developed using chemiluminescence (PIERCE) and visualized with a PhosphoImager (BioRad, Hercules, CA), and bands were quantified using QuantityOne software (BioRad).
Dose Response Data Analysis:
Neutral control condition (DMSO) was included.
Corrected SCNA was calculated as: Density of SNCA at dose / Density of Actin at dose
Percent activation of SNCA expression was calculated as follows:
100 * ((Corrected SNCA at dose/ Corrected SCNA at neutral) - 1)
ECMax was determined to be the dose that supported Maximum Observed Activity.
Inactive compounds = 0
Active compounds = -10 * Log(ECMax)
Activity_Outcome = 1 (inactive)
Dose Dependent Activity is not observed.
Activity_Outcome = 2 (active)
Dose Dependent Activity is observed.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)