Goat anti mouse horseradish peroxidase

Anti-α-synuclein antibody (BD Transduction Laboratories, 610786), anti-phosphorylated pS129 α-synuclein antibody (ab184674, Abcam), anti-β-actin antibody (Abcam, ab8227), anti-HA antibody (Roche, 118674231001), anti-TH antibodies (BD Transduction Laboratories, 612300, for immunoblotting and immunohistochemistry; Novus, NB300-109, for immunocytochemistry), anti-Iba-1 antibody (019-19741, Wako), anti-GABA_A receptor β2/3 antibody (Millipore, 05-474), anti-HSP90 antibody (BD Transduction Laboratories, 610418), anti-14-3-3 antibody (Millipore, 06-511), MG132 (Sigma, C2211), MPP+ iodide (Sigma, D048), MPTP hydrochloride (Sigma, M0896), normal goat serum (G9023, Sigma-Aldrich), goat anti-rabbit horseradish peroxidase (111-035-144, Jackson ImmunoResearch), goat anti-mouse horseradish peroxidase (115-035-146, Jackson ImmunoResearch). Tat-βsyn-degron peptide (YGRKKRRQRRRRTKSGVYLVGRRRG), Tat-βsynN-degron peptide (YGRKKRRQRRRGVLYVGSKTRRRRG), and Tat-βsyn control peptide (YGRKKRRQRRRRTKSGVYLVG) were chemically synthesized by GL Biochem (Shanghai, China). Tat peptide (YGRKKRRQRRR) was synthesized in our lab using the Prelude peptide synthesizer (Protein Technologies Inc.).

DNA-free total RNA was extracted from LV samples by a standard protocol with an EZ-RNA-total RNA Isolation Kit (Biological Industries, Israel). cDNA synthesis (500 ng) was carried out with the Verso RT-PCR Kit (ABgene, USA) according to the manufacturer’s instructions. qRT-PCR was performed on a Bio-Rad iQ-Cycler using a SYBR Green PCR kit (Invitrogen, Israel). The primer sequences are provided in S1 Data. Western blot was performed according to standard procedure [3 (link)] using the following antibodies: mouse monoclonal anti-Cytochrome C (Cyt C) (Cat # ab14734, Abcam, USA), mouse monoclonal anti-Voltage-dependent anion channel (VDAC)1/porin (Cat # ab110325, Abcam, USA), and mouse monoclonal anti- sirtulin (Sirt)3 (Cat # Sc-365175, Santa Cruz Biotechnology, USA), rabbit anti-GAPDH (Cat # 37168, Abcam, USA) followed by goat anti-mouse horseradish peroxidase (Cat # 10015289, Jackson ImmunoResearch Laboratories, Inc. USA) and goat anti-rabbit horseradish peroxidase (Cat # 111035003, Jackson ImmunoResearch Laboratories, Inc. USA)

Estrogen receptor α, cMyc, and α-tubulin protein levels were determined using immunoblotting with mouse anti-human ERα antibody (clone 6F11, Novocastra Laboratories, UK), mouse anti-human cMyc antibody (9e10, Abcam, MA, USA), and mouse anti-human α-tubulin antibody (clone DM-1A, Sigma-Aldrich, MO, USA), respectively. Goat anti-mouse horseradish peroxidase and alkaline phosphatase were used as secondary antibodies (Jackson ImmunoResearch Laboratories, PA, USA). Densitometric analyses were performed using Quantity One 4.6 (Bio-Rad Laboratories, CA, USA). The changes in the expression due to treatment with the ER-targeted probes were performed in cells grown in phenol red-free medium for a minimum of 5 days prior to the treatment. The expression of ER in the different human breast cancer cell lines was quantified by normalizing the intensity of the bands to those of standard, known concentrations of recombinant hERα protein (PanVera, Inc., Madison, WI, USA).