Journal: Frontiers in Molecular Neuroscience

Article Title: Pathophysiological Consequences of Neuronal α-Synuclein Overexpression: Impacts on Ion Homeostasis, Stress Signaling, Mitochondrial Integrity, and Electrical Activity

doi: 10.3389/fnmol.2018.00049

Figure Lengend Snippet: Impact of α-Syn overexpression on stress kinases, caspases, and apoptosis-related proteins. Western blots are shown for cell lysates isolated from cortical neurons at 12 days of synuclein overexpression under denaturing conditions and electrophoresed on SDS-PAGE. (A) Expression analysis of AAV-expressed rat and human α-Syn at different AAV titres and Bcl-Xl (at 3x10e7 tu) to delineate the level of overexpression (see Materials and Methods for more details). (B) Detection of levels of phosphorylated JNK (panel 1), phosphorylated p38 (panel 2) and phosphorylated Erk (panel 3; antibody CS4377) stress kinases, detection of non-phosphorylated Erk is shown in panel 4 as loading control. (C) Detection of the levels of mitochondrial proteins Tom-20 (panel 1) and VDAC-1 (panel 2), and lysosomal proteins LC3B1 and −2 (panel 3). (D) Detection of levels of cleaved caspase 3 (panel 1), cleaved caspase 9 (panel 2), and phospho-Erk (panel 3; antibody CS4376). (E) Detection of phospho-Bad in a high molecular weight complex. (F) Detection of phosphorylated α-Syn (S129). (G) Detection of Bim and Akt, Tubulin and EGFP (which is co-expressed with the synucleins) as loading controls. EGFP, neurons transduced with AAV-EGFP (1 × 10e8 tu); α-S, neurons transduced with AAV-α-Syn-EGFP (expressing α-Syn and EGFP from independent transcription units, 1 × 10e8 tu). ßS, neurons transduced with AAV-ß-Syn-EGFP (1 × 10e8 tu); γ-S, neurons transduced with AAV-γ-Syn-EGFP(1 × 10e8 tu); Bcl-Xl, neurons transduced additionally with AAV-Bcl-Xl (3 × 10e7 tu).

Article Snippet: Protein lysates obtained from cultured neurons were analyzed by denaturing SDS-PAGE (20 μg protein / lane), blotting to nitrocellulose or PVDF membranes and incubation with the following primary antibodies: rat α-Syn, Abcam ab87599 or CST #4179; human α-syn, Invitrogen 32-8100; phospho S129 α-Syn Abcam ab168381; cleaved caspase 3, CST #9661; cleaved caspase 9, CST #9507; phospho-p54/p46 SAPK/JNK, CST #4668; phospho-p38 MAPK, CST #9216; phospho-p44/p42 ERk1/2, CST #4376 or CST#4377; p44/p42 Erk1/2, CST # 9102; Tom20, SantaCruz 11413; VDAC-1, Abcam 15895; LC3B-I / -II, CST #2775; Bim-1, CST # 2933; phospho-AKT, CST #9271; Akt, CST #9272; Bcl-Xl, CST #2764; phospho-Bad (S136), CST #9295; c-Ret (C-20), SantaCruz 1290; TrkA, CST #2508; TrkB, CST #4603; TrkC, CST #3376; EGFP, Roche 11814460001; ß-Tubulin, Sigma, T-4026; Chemoluminescent detection was performed on X-ray film or a BioRad ChemiDoc XRS+ Imager.

Techniques: Over Expression, Western Blot, Isolation, SDS Page, Expressing, Molecular Weight, Transduction