Gradient sds gel

HEK293 cells were grown in 100 mm plates and transiently transfected with 5 μg of HA-hARGLU1 together with 5 μg of either FLAG-hPUF60, FLAG-hU2AF2 or FLAG-hJMJD6. Forty-eight hours later, cells were harvested and protein was extracted. Protein lysates were pre-cleared with protein G beads, split into three tubes and incubated with 5 μg of anti-FLAG (1:1000; Sigma, F1804) or anti-HA (1:1000; Cell Signaling, 3724S) antibody. The next day, 50 μl of protein G agarose was added to the lysates and incubated on a rotator for 3–4 h at 4°C. Proteins were eluted in lithium dodecyl sulfate buffer (LDS, Invitrogen) and resolved on a 4–20% gradient SDS gel (Bio-Rad, Hercules, CA, USA).

Immunoblot analysis was performed as described previously [8 (link)]. In short, all lysates for immunoblot analysis were prepared by lysing cells in PBS‐1%TritonX‐100 buffer supplemented with Protease Inhibitor Cocktail and Phosphatase inhibitor (Roche, Basel, Switzerland). After that, the resulting suspension was incubated on ice for 10 min, centrifuged, and the supernatant collected. Tissue lysates were made in the mentioned lysis buffer using Precellys 24 tissue homogenizer (Bertin instruments) and the Precellys kit (#KT03961‐1‐003.2). Proteins were separated on gradient SDS gels (BioRad) and transferred to nitrocellulose membranes (BioRad #1704158). Transfer quality was evaluated with Ponceau staining (Sigma, #P7170). Immunoblots were quantitated using image j (Bethesda, MD, USA). Statistical significance was determined via a one‐sided t‐test, unless specified otherwise in the figure legends.

Cells at 60–70% confluency were treated as indicated in the results section. Cells were washed in PBS and scraped in a 100 µL RIPA lysis buffer containing protease inhibitor cOmplete (Roche, Mannheim, Germany), PMSF (1 mM), and orthovanadate (1 mM). Total protein was quantified using a DC™ protein assay (Bio-Rad, Hercules, CA, USA). A total of 15 µg of proteins was separated using gradient SDS gels (4–20%, Bio-Rad) and blotted on nitrocellulose membranes by a Turbo Blot (Bio-Rad). Gene signals were detected as described before [23 (link)].
uPAR protein levels were determined by ELISA (DUP00, R&D Systems, Minneapolis, USA) according to the manufacturer’s protocol. In brief, cell lysates from 10⁵ to 10⁶ cells were 10-fold diluted in a RIPA lysis buffer, and 50 μL of cell lysates or standard was added to 100 μL of assay diluent RD1W solution. The samples were incubated for two hours at RT and washed four times with a 400 μL wash buffer. A total of 200 μL of human uPAR conjugate was added and incubated for 2 h at RT. After four washing steps, 200 μL of substrate solution was added and incubated for 30 min at RT protected from light before adding 50 μL of stop solution. The optical density was measured at 450 nm with a reference of 540 nm on a Tecan reader Infinite 200 Pro. uPAR concentrations were calculated for 10⁶ cells.