Mg132

The hiPSCs were cultured on Matrigel growth factor-reduced basement membrane matrix (Corning) in mTeSR medium (STEMCELL Technologies). The cells were treated with rapamycin (200 nM–2 μM, Fisher Scientific) or DMSO for indicated periods and thereafter tested for reversion in the absence of rapamycin. To analyze the proteasome function, hiPSCs were treated with 100 nM MG132 (proteasomal inhibitor, Thermo Scientific; cat. no. 508339) for 24 h. Information regarding cell lines is given in the supplemental experimental procedures.

Chemical reagents used in this study and their commercial sources were as follows: dydrogesterone (HY-B0257A), lumacaftor (HY-13262), abiraterone acetate (HY-75054), eltrombopag (HY-15306), ethinylestradiol (HY-B0216), and nafamostat (HY-B0190) were from MedChemExpress (Monmouth Junction, NJ, USA). Methyltestosterone (M1800000), quinestrol (E7887), nandrolone phenpropionate (BP260), and testosterone propionate (T1875) were from MERCK (Darmstadt, Germany). 25-Hydroxycholesterol (sc-214091) was from Santa Cruz Biotechnology (Dallas, TX, USA). Compounds were solubilized in DMSO and used at a concentration of 10 µM for 48 h. The proteasome inhibitor MG132 (Thermo Fisher Scientific, Waltham, MA, USA) was used at 10 µM for 24 h.

SK-MEL-28 and SK-MEL-29 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and from Sloan-Kettering Memorial Center, respectively, and propagated in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The human neuroblastoma cell line SH-SY5Y over expressing α-syn (SH/αS) and control SH-SY5Y cells were a kind gift of Dr. Joseph R Mazzulli (Northwestern University) and propagated in Opti-MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. CRISPR/Cas9 genome editing was used to target SNCA in SK-MEL-29 cells as described previously^{31 (link)} for SK-MEL-28 cells using α-syn CRISPR/Cas9 knockout plasmid (Santa Cruz Biotechnology # sc-417273-NIC). Lentivirus particles expressing human α-syn under cytomegalovirus (CMV) promoter (Applied Biological Materials, Inc, Canada) was used to re-express α-syn in SK-MEL-28 KO cells as described previously^{31 (link)}. For proteasome and autophagy inhibition experiments cells were treated with 10 µM MG132 (Thermofisher Scientific, # M7449) for 6 h and 50 nM bafilomycin A1 (Millipore, # B1793) for 5 h, respectively, in growth medium. The cell lines were authenticated and tested for mycoplasma contamination using MycoAlert® Mycoplasma Detection Kit (# LT07-318).

β‐arrestin1 and β‐arrestin2 siRNA (50 nM) or nontargeting siRNA (50 nM) were transfected into HUVECs for 24 h and the proteasome inhibitor MG132 (0.2 µM; Thermo Fisher Scientific) was added to the HUVECs for 48 h. The HUVECs were washed with PBS and lysed in NP‐40 lysis buffer (Boston Bioproducts) including protease and phosphatase inhibitor (Roche). The cell lysates were incubated rotated with rabbit anti‐eNOS antibody (CST; 1:50) at 4°C for overnight. Protein A/G agarose beads (Santa‐Cruz) were added to immunoprecipitated samples and rotated for 2 h with rotation at 4°C. After twice washes in lysis buffer and once in PBS at 4°C, the agarose beads were boiled in Laemmli SDS sample buffer (Alfa Aesar) for western blot analysis with rabbit anti‐eNOS antibody (CST; 1:1000), ubiquitin antibody (Santa‐Cruz; 1:200), β‐arrestin antibody (CST; 1:1000), and GAPDH antibody (CST; 1:1000).