Odyssey xf imager

Western blot assays were performed as described previously^{13 (link)}. In brief, cells or purified mitochondria were resuspended in RIPA buffer (TEKnova) containing 1% protease inhibitor cocktail (Sigma). Protein concentration was determined using BCA protein assay (Pierce). To analyze mitochondrial proteins, lysates were heated at 42 °C for 5 min in Laemmli sample buffer (Bio-Rad), but proteins from whole cell lysate were heated at 95 °C for 5 min in the same buffer. 10–30 µg protein lysates were loaded onto 4–20% gradient SDS PAGE (Bio-Rad) and transferred to PVDF membranes (Roche). Membranes were probed with the primary antibodies followed by washing and adding the fluorescent secondary antibodies. Antibody-antigen reaction profiles were visualized and quantified by Odyssey XF Imager (LI-COR). Antibodies are summarized in Supplementary Table 3.

Cells were washed one time with PBS before pelleting. Pellets were resuspended in 1x RIPA buffer with protease and phosphatase inhibitors added. After shaking samples at 4°C at 600 rpm, samples were centrifuged for 10 minutes at 14,000xg. The lysates were then quantified using Bradford assay and diluted to 1x LDS. Samples were loaded on a 4–12% Bis-Tris gel (ThermoFisher, NP0321BOX) and run in MOPS Running Buffer (ThermoFisher, NP0001) followed by transfer to PVDF membranes using a TransBlot Turbo Transfer System at 25 V, 1.3 A, for 12 min (Bio-Rad). Total protein was quantified using total protein stain (LI-COR, 926–11011) and blocked in 5% milk in 1x TBST. Blot was incubated overnight with primary antibody in 1x TBST with 5% BSA at 4°C. Blots were then washed in 1x TBST, incubated in secondary antibody for 1 hour at room temperature in 5% milk, and washed three times for 10 minutes. Blots were incubated with HRP substrate and imaged on the LI-COR Odyssey XF Imager.

SDS–PAGE was done using homemade Bis-acrylamide gels or Mini-PROTEAN TGX gradient gels (Bio-Rad). Gels were blotted onto PVDF membranes. Membranes were blocked in 5% milk powder or 10% Roti-Block (Roth). Primary antibodies were added overnight at 4°C in 5% milk powder or 10% Roti-Block (Roth)—1:1,000 Ub antibody P4D4 (Santa Cruz Biotechnology), 1:3,000 Hip1 (22231-1AP; Proteintech), 1:000 PARP10/ARD10 (NB100-2157; Novus Biologicals), 1:1,000 Rad23b (E-AB-62188; Elabscience), 1:1,000 Dsk2 (ab4119-100; Abcam), 1:4,000 Rad23 (Sommer Lab), 1:1,000 Epn2 (Invitrogen), DDI2 (AB197081; Abcam), 1:1,000 DDI1 serum (Jeffrey Gerst, Weizmann Institute of Science (99 (link))), 1:1,000 APPL1 (D83H4; Cell Signaling), 1:1,000 CCDC50 (AB127169; Abcam), 1:1,000 FAF1 (1027-1-AP; Proteintech), 1:1,000 ZFAND2B (Sigma-Aldrich), 1:1,000 Riok3 (13593-1-AP; Proteintech), 1:1,000 USP11 (22340-1-AP; Proteintech), 1:5,000 CDC48 (Sommer Lab), 1:1,000 Vps9 (Scott Emr, Cornell University (100 (link))), 1:1000 YUH1 serum (Tingting Yao, Colorado State (101 (link))). Anti-mouse IgG HRP (Sigma-Aldrich) and anti-rabbit IgG HRP (Sigma-Aldrich) were used at 1:10,000 as secondary antibodies. Immunoblots were visualized using an Odyssey XF imager (LI-COR).

1.25 μg ub chains were incubated with 1 μM DUB, either K48-specific OTUB1 or K63-specific AMSH, in disassembly buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 10 mM DTT) for 45 min at 37°C. Reactions were stopped with SDS–DTT sample buffer. Samples were run on SDS–PAGE and Western blot with anti-Ub antibody and imaged using an Odyssey XF imager (LI-COR).

First, 1.5 × 10⁵ HEK293H cells were seeded in each cavity of a 24-well plate one day prior to transfection with the pDESTsplice reporter constructs. Cells were transfected using Lipofectamine LTX Reagent according to the manufacturer’s instructions. After 4 h, a medium change was performed. After a total of 48 h post-transfection in culture, cells were washed with PBS, dissolved in RNA Lysis Buffer, and RNA was isolated using the associated Quick-RNA Miniprep Kit (Zymo Research, Freiburg i. Breisgau, Germany). RNA concentration was determined in a Spark Microplate Reader (Tecan, Männedorf, Switzerland) equipped with a NanoQuant Plate. Subsequently, 500 ng of the obtained RNA was transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Braunschweig, Germany). Polymerase chain reaction was performed using DreamTaq DNA Polymerase Mastermix (Thermo Fisher, Braunschweig, Germany) with the addition of the template cDNA and the desired primer pair (Table S3). The PCR result was applied to a 1.5–2% TAE agarose gel to which Sybr Safe (Thermo Fisher, Braunschweig, Germany) was added as DNA band staining. Bands were captured at 600 nm in the Odyssey XF imager (LI-COR Biosciences, Bad Homburg v. d. Höhe, Germany) and analyzed using Empiria Studio 2.2.