Anti v5 antibody r960 25

Ubiquitination assay was performed as described elsewhere [20 (link)]. In brief, HEK293 cells (ATCC) were transfected with plasmids expressing HA-tagged Ub, V5-tagged Nrf2, and FLAG-tagged Keap1 [21 (link)] for 48 h and then treated with MGS for 16 h. Prior to lysis, cells were treated with 10 μM of MG132 (Sigma-Aldrich) for 3 h to block ubiquitin-dependent protein degradation. The total cell lysate was prepared by Pierce™ IP lysis buffer per the manufacturer’s protocols (Thermo Scientific). For precipitating V5-tagged Nrf2, 1 μg of anti-V5 antibody (R960–25, Thermo Scientific) was added to the cytosolic fraction. Immune complexes captured by protein A-sepharose (Thermo Scientific) were analyzed by immunoblotting for HA (H3663, Sigma) to reveal the ubiquitinated Nrf2.

Western blotting was performed using antibodies against STT3A (12034-1-AP), STT3B (15323-1-AP), MAGT1 (17430-1-AP), and prosaposin (10801-1-AP) from the Proteintech Group (Rosemont, IL). Antibodies against TUSC3 (SAB4503183) and actin (A5316) were purchased from Sigma-Aldrich (St. Louis, MO). The antibody against EMC3 (sc-365903) was purchased from Santa Cruz Biotechnology (Dallas, TX). The antibody against SHBG (MAB2656) was purchased from R&D Systems (Minneapolis, MN). The antibody against HA (C29F4) was acquired from Cell Signaling Technology (Danvers, MA). The anti-V5 antibody (R960-25) was purchased from Thermo Fisher Scientific. The anti-NS1 monoclonal antibody 1F11 was a gift from P. Malasit at the National Center for Genetic Engineering and Biotechnology in Thailand.

Surface nuclear spreads were performed as described previously (Jin et al, 2009 (link)). In brief, yeast cells enriched at prophase I (∼5 h after induction of meiosis) were spheroplasted by lyticase treatment. Spheroplasts were then fixed and poured onto a glass slide. The slide was then rinsed with PhotoFlo 200 and air-dried, followed by PBS buffer with 3% BSA to block for 2 h at room temperature. Anti-V5 antibody (R960-25; Thermo Fisher Scientific) was used to detect V5-Csm4 and Ndj1-V5; anti-HA antibody (12CA5; Roche/Sigma-Aldrich) was used to detect Mps2-3HA. Rec8-GFP was detected by an anti-GFP mouse monoclonal antibody (ab209; Abcam). Secondary antibodies (FITC-conjugated goat antirabbit, rhodamine-conjugated goat antimouse, and Cy3-conjugated goat antirat; Jackson ImmunoResearch Laboratories) were used at a dilution of 1:500. Mounting medium with DAPI was added before microscopy. Images were acquired with an epifluorescence microscope (Axio Imager M1; Zeiss) with a 100× objective lens (NA = 1.40) at room temperature.