Ubiqapture q kit

The ubiquitination level of protein was measured by a UbiQapture-Q kit (Enzo, Switzerland) according to the manufacturer’s instructions. After injection with 100 μL of MG132 and PR-619 or equivalent DMSO, mud crab hemocytes were collected and homogenized with cell lysis buffer for Western blotting and IP (Beyotime, China). Then, 40 μL of UbiQapture-Q matrix was added to the cell lysate. Subsequently, the mixture was resuspended gently with the affinity matrix at 4°C overnight. After centrifugation at 5,000 × g for 15 s, the matrix was washed with PBS and subjected to Western blotting. Similarly, S2 cells transfected with the indicated plasmids were lysed and used for ubiquitination assay.

Immunoprecipitation of ubiquitinylated proteins was performed using the UbiQapture-Q Kit (Enzo Life Sciences), following the manufacturer’s instructions. Briefly, protein samples were harvested from BMDM and protein concentration was measured by BCA assay. Ubiquitinylated proteins were captured from 25 μg of total proteins lysates using 40 μl UbiQapture-Q matrix. Captured and uncaptured fraction samples were analyzed by Western blotting, as detailed above.

The ubiquitinated proteins were isolated using the UBIQAPTURE-Q® kit (UW8995, Enzo Life Sciences). KRAS protein activity was examined by the KRAS activation assay kit (ab211159, Abcam) following company’s instruction.

Ubiquitylated proteins were isolated using the UbiQapture-Q kit (Enzo Life Sciences, Farmingdale, New York, USA) according to the manufacturer’s instructions. Ubiquitylated proteins were isolated from a total of 1 × 10⁷ cells lysed with TEN buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100), supplemented with 100 mM N-ethylmaleimide (NEM) to inhibit deubiquitinase activity [35 ]. A total of 40 μl of UbiQapture-Q matrix was pre-equilibrated in TEN buffer and incubated with cell lysates (200 μl) by rotating at 4˚C overnight. After washing five times, captured proteins were eluted with 2x SDS-PAGE sample buffer containing 10mM DTT. Samples were resolved in 4–12% acrylamide gels, transferred onto PVDF membranes and analyzed by Western blotting using anti-HA antibody in blocking buffer (TBST supplemented with 1% BSA).

Western blot experiments were performed with cell lysates harvested at different time points after ATO treatment. All the proteins were loaded into each well of a 15% SDS-PAGE after boiling. Gels were transferred onto PVDF membranes (Bio-Rad), blocked with 5% milk/PBS, and incubated overnight at 4 °C with primary antibodies. Following washing and incubation with secondary antibodies in 5% milk, the membrane was washed, and the positive signals were developed with chemiluminescence reagent (Amersham™). The immunoprecipitation assays were carried out with using Pierce™ IP lysis buffer (Thermo), protein A-agarose beads (Invitrogen), Ubiqapture-Q^® kit (ENZO life sciences). For immunoprecipitation, 2 µl Mouse anti-PML mAb, 20 µl of 50% slurry of protein G beads were added to each samples incubated at 4 ℃ for 16 h at a rotator in the immunoprecipitation experiment. Beads were washed four times with 1 ml of ten-fold diluted urea buffer and 20 µl of 2 × SDS loading buffer was added. Immunoprecipitations were analyzed by western blot as indicated. Antibodies: mouse anti PML antibody (Millipore Sigma, P6746), rabbit anti YAP antibody (CST, 14074), rabbit anti beta-actin antibody (Proteintech, 60008-1-Ig), HRP conjugated anti IgG secondary antibodies (Proteintech).

Ubiquitinylation of CXCR4 was evaluated by two different methods. Jurkat cells co- transfected with GFP and Nef or null plasmids were evaluated by flow cytometry for Nef induced CD4 downregulation. After adjusting to constant levels of GFP expression, cells were disrupted and ubiquitinylated proteins were recovered using UbiQapture™-Q Kit, containing a high-binding affinity matrix of immobilized monoclonal anti-ubiquitin antibodies (ENZO Life Sciences, Germany), following manufacturer's instructions. Ubiquitinylated proteins were resolved by SDS/PAGE and CXCR4 was detected by immuno-blotting using a monoclonal antibody (Abcam). Alternatively, Nef (+) or Nef (−) transfectants co-expressing FLAG-tagged ubiquitin and HA tagged CXCR4 were lysed using the lysis buffer (150 mM NaCl, 50 mM Tris, 1% NP40, and protease inhibitor cocktail complete (Roche Diagnostics)) followed by immuno-precipitation of ubiquitinated proteins using M2 FLAG mAb agarose (Sigma Aldrich, St. Louis, MO) and immuno-blotting with anti-HA (Roche Diagnostics) to detect the ubiquitinylated receptor.