The Signal-Seeker Ubiquitin Enrichment Kit (BK161, Cytoskeleton) was used according to the manufacturer’s instructions to pull down ubiquitinated proteins. Briefly, H1975 cells with or without MTSS1 overexpression were collected and lysed with BlastR™ lysis buffer with inhibitors (de-ubiquitinase inhibitor, Cat # NEM09BB; protease inhibitor cocktail, Cat # PIC02). The lysates were transferred into BlastR™ filters and diluted with BlastR™ dilution buffer to the final volume. Equal amounts of protein were incubated with 20 µL ubiquitination affinity beads or control beads for 2 h at 4 °C. The beads were washed 3 times with BlastR-2™ wash buffer, followed by incubation with 30 µL elution buffer for 5 min at room temperature. The precipitates were collected by the spin columns provided in the kit and were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
For His-tagged ubiquitin pulldown with Ni–NTA beads, HeLa cells were transfected with wild-type (His–Ubiquitin–WT) or lysine-mutated (His–Ubiquitin–KO) ubiquitin and the other plasmids for 60–72 h. Cells were harvested and resuspended in Buffer A (6 M guanidine–HCl, 0.1 M Na2HPO4/NaH2PO4, 10 mM imidazole pH 8.0) with inhibitors (10 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 5 mM N-Ethylmaleimide and Protease inhibitor cocktail). The lysates were sonicated (75 W, 2 s, 5 s, 3 min) before mixing with Ni–NTA beads (QIAGEN) by rotating at room temperature for 3 h. Subsequently, the His pull-down products were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and once with buffer TI (25 mM Tris-HCl and 20 mM imidazole, pH 6.8). Then elution buffer (0.2 M imidazole, 5% w/v SDS, 0.15 M Tris-Cl, pH 6.8) was added and incubated for 20 min at room temperature. The supernatants were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
For Flag-tagged ubiquitin pulldown, 293 T or HeLa cells transfected with Flag-ubiquitin were lysed with IP buffer with inhibitors (10 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 5 mM N-Ethylmaleimide and Protease inhibitor cocktail). The subsequent immunoprecipitation was performed as described above. For PD-L1 deglycosylation, immunoprecipitates or whole cell lysates were treated using PNGase F according to the manufacturer’s instructions. Finally, samples were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
For His-tagged ubiquitin pulldown with Ni–NTA beads, HeLa cells were transfected with wild-type (His–Ubiquitin–WT) or lysine-mutated (His–Ubiquitin–KO) ubiquitin and the other plasmids for 60–72 h. Cells were harvested and resuspended in Buffer A (6 M guanidine–HCl, 0.1 M Na2HPO4/NaH2PO4, 10 mM imidazole pH 8.0) with inhibitors (10 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 5 mM N-Ethylmaleimide and Protease inhibitor cocktail). The lysates were sonicated (75 W, 2 s, 5 s, 3 min) before mixing with Ni–NTA beads (QIAGEN) by rotating at room temperature for 3 h. Subsequently, the His pull-down products were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and once with buffer TI (25 mM Tris-HCl and 20 mM imidazole, pH 6.8). Then elution buffer (0.2 M imidazole, 5% w/v SDS, 0.15 M Tris-Cl, pH 6.8) was added and incubated for 20 min at room temperature. The supernatants were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
For Flag-tagged ubiquitin pulldown, 293 T or HeLa cells transfected with Flag-ubiquitin were lysed with IP buffer with inhibitors (10 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 5 mM N-Ethylmaleimide and Protease inhibitor cocktail). The subsequent immunoprecipitation was performed as described above. For PD-L1 deglycosylation, immunoprecipitates or whole cell lysates were treated using PNGase F according to the manufacturer’s instructions. Finally, samples were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
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