Mg132

Cells were seeded in a 96-well plate with 10,000 cells per well in 200 μl complete growth medium. Eight hours prior to cell death initiation, medium was exchanged for Complete DMEM with DOX (1 μg/ml) or DMSO. Cells were subsequently treated with MG132 (4 μM, APExBio). Imaging was subsequently performed using the IncuCyte S3 (Sartorius, Göttingen, Germany; version 2021C). Nine images per well were captured, analyzed, and averaged. Cell death was assessed through measuring uptake of YoYo-1 (50 nM, Thermo Fisher Scientific) and expressed as the area of YoYo-1⁺ cells as a percentage of the total phase area. In experiments where zVAD-fmk and GSK’843 were used, zVAD-fmk (20 μM, APExBio) was administered 30 minutes prior to treatment with GSK’843 (20 μM, Sigma) and MG132.

Antibodies specific for c-Met (#25869-1-AP); c-Cbl (#25818-1-AP); Fibronectin (#1G10F9); Vimentin (#10366-1-AP); E-cadherin (#20874-1-AP); Tubulin (#10094-1-AP); DYKDDDDK (#66008-3-Ig); β-actin (#66009-1-Ig); mTOR (#20657-1-AP) were obtained from proteintech (Wuhan, China). The Phospho-AKT (Ser473) (#4060) and AKT (#9272) antibodies were both supplied by Cell Signaling Technology (Danvers, MA). An anti-phosphotyrosine antibody was obtained from Abbkine. Antibodies used were goat polyclonal to ORP5 (Abcam, ab59016); mouse monoclonal to N-cadherin (Servicebio, GB12135); p-mTOR (59. Ser 2448) (Santa Cruz Biotechnology, sc-293133). For immunoblotting, horseradish peroxidase-conjugated secondary antibodies were obtained from Beyotime. For immunofluorescence, CoraLite488––conjugated Affinipure Goat Anti-Mouse IgG(H + L) was obtained from proteintech.
Chloroquine Sulfate and MG-132 were from APExBIO (MA, USA). Cycloheximide (CHX) was from MedChemExpress (shanghai, China).

The stability of GFP-tagged α-arrestins expressed from the pRS415-TEF1 promoter plasmids (see Supplemental Table S2) was assessed by immunoblotting. Cells were grown to mid-exponential growth phase (A₆₀₀ 0.8–1.0) at 30 °C and cells were treated with 0.1 mg/mL CHX (Gold Bio, St. Louis, MD, USA) to block new protein synthesis; equal densities of cells were harvested over time. Cell pellets were treated as indicated above in Section 2.5, and TCA whole-cell protein extracts were made, resolved by SDS-PAGE, and the GFP-tagged proteins were detected by immunoblotting. To determine the impact of proteasomal inhibition on α-arrestin protein stability, similar assays were performed in cells where the multi-drug resistance pump, Pdr5, was deleted. Cells were then treated with 100 μM MG132, a proteasomal inhibitor (APExBIO, Houston, TX, USA), for 1 h prior to the addition of 0.1 mg/mL CHX. Once again, equal densities of cells were harvested at time points post-treatment with MG132 and cycloheximide. TCA extracts were generated, proteins were resolved by SDS-PAGE and identified via immunoblotting. Quantification of proteins on membrane blots was performed using Image J software, as described in Section 2.5 above.

After 48 h of transfection, cells were treated with 5 µg/mL actinomycin D (ActD; APExBIO, Houston, TX, USA) to inhibit RNA synthesis and harvested at 0, 0.5, 1, 2, and 4 h. RNA was extracted and quantified by qRT-PCR to detect mRNA stability. To determine protein stability, cycloheximide (CHX; 20 µg/mL; APExBIO) was added to the culture medium to inhibit protein synthesis for 0, 2, 4, and 8 h, and protein was extracted and detected by western blotting. At 48 h after transfection, cells were starved overnight and exposed to 1 µg/mL MG132 (APExBIO) for 6 h to inhibit protein degradation, and protein was extracted for western blotting.