Dynabeads m 280 anti mouse igg

Chromosomal DNA replication was analyzed based on bromodeoxyuridine (BrdU) incorporation as previously described^{26 (link),27 (link)}, with some modifications. Derivatives of the cdc25-22 Pnmt1-TK Padh1-hENT strain expressing the herpes simplex virus thymidine kinase (TK) gene and the human equilibrative nucleoside transporter (ENT) gene were grown in Edinburgh minimal medium (EMM) at 25 °C to induce TK expression and incubated at 35.5 °C for 4 h for G₂/M arrest. (Note that the trt1∆ strains were grown in YES medium to maintain their cell growth.) Cells were released at 25 °C to resume cell cycle progression in the presence of 200 µM BrdU (Sigma-Aldrich) with or without 10 mM HU (Sigma-Aldrich) and were fixed with 0.1% sodium azide at pre-determined times. Genomic DNA was prepared by vigorously vortexing of the cells in S&G buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1% SDS, 2% Triton X-100) in the presence of phenol/chloroform, followed by ethanol precipitation. The DNA was fragmented by sonication and heat-denatured, and then used for immunoprecipitation with anti-BrdU antibody (3D4; Becton Dickinson/Pharmingen) bound to Dynabeads M-280 anti-mouse IgG (Thermo Fisher). The DNA was recovered by treatment with 1% SDS and 250 µg/ml proteinase K, and was evaluated by quantitative PCR (qPCR) with the primers listed in Supplemental Table S2.

Whole cell protein lysates were prepared from 90% confluent cell cultures in 100 mm culture dishes (Sigma, St. Louis, MO). Briefly, cell pellets were resuspended in 100 mM 5,5-dimethylcyclohexane-1,3-dione (Dimedone, Sigma) and incubated at 37°C for 60 min under agitation. After washing with PBS, pellets were lysed in RIPA buffer (Cell Signaling) and then sonicated for 15 seconds using Sonic Dismembrator (ThermoFisher). Following sonication, supernatant was moved to a new tube and samples were incubated with 2 μg/mL primary antibody [mouse anti-Keap1, 1:100 (Abcam)] for 24 h at 4°C. Magnetic beads [Dynabeads M-280 anti-mouse IgG (Thermo Fisher Scientific)] was then added and samples were incubated for 3 hours at 4°C. After 3 washes, samples were analyzed by Western blotting as described above.
For co-immunoprecipitation of Cav-1/Keap1/Nrf2 complex, a direct immunoprecipitation method was used. Briefly, magnetic beads [Dynabeads M-280 anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific)] were incubated with primary antibody [anti-rabbit Nrf2 1:100 (Santa Cruz) or anti-mouse Keap1 1:100 (Abcam)]) for 6 h at 4°C. Beads were then washed to remove unbound primary antibody. Following treatment (400 μM H₂O₂, 1 h), cell lysates were added to the Dynabead/Antibody cocktail and incubated at 4°C overnight. Samples were washed twice with PBS and then analyzed by Western blot.

ChIP experiments followed a protocol adapted and modified from Kimura et al. (2008) (link). At least 5×10⁶ human cells (or 50×10⁶ chicken DT40 cells) were used per each ChIP experiment, crosslinked with 1% formaldehyde (Sigma-Aldrich, St Louis, MO) for 5 min at room temperature. Crosslinked chromatin was snap-frozen, and samples from human cells were sheared by sonication. Samples from chicken DT40 cells were instead digested with 200 U/ml of Micrococcal nuclease (Worthington Biochem. Corp.) for 30 min at 21°C, and sonicated briefly. Immunoprecipitation was performed with anti-mouse IgG Dynabeads M-280 (Life Technologies, Carlsbad, CA) conjugated with primary antibodies (see above), using 10⁶ cells (10×10⁶ cells for chicken) each. Samples were decrosslinked at 93–100°C for 12 min, and treated with RNase A and proteinase K, and DNA was purified with Chelex beads (Bio-Rad).
To quantify the IP DNA, qPCR was performed on Input and IP samples using a SYBR Green master mix (Roche, Penzberg, Germany). Primers were used at 400 nM and are described in Table S1. Percentage of recovered IP material was calculated relative to standard curves calculated from Input using the second derivative maximum algorithm in the LightCycler 480 software, to account for differential primer efficiency.