Prometheus prosignal dura

After entrainment, seedlings were exposed to constant darkness, constant 60 μmol m⁻² s⁻¹ monochromatic blue or red light under LEDs (XtremeLUX, Santa Clara, CA), or 50–60 μmol m⁻² s⁻¹ white light at 22°C. Seedlings were moved at dawn (Time 0) and collected every 4 h from Times 0 to 48 (RVE4‐FLAG) or every 3 h from 3 h before dawn to Time 33 (RVE8‐HA). Samples were prepared and quantified as previously described (Shalit‐Kaneh et al., 2018 (link)). Total protein was analyzed by western blotting using mouse monoclonal anti‐FLAG M2‐HRP antibody (Sigma‐Aldrich, St. Louis, MO) for RVE4‐FLAG and rat monoclonal anti‐HA‐HRP antibody (Roche, Basel, Switzerland) for RVE8‐HA. Prometheus ProSignal Dura (Genesee Scientific, Rochester, NY) was used to generate peroxidase activity and a Chemidoc analyzer (Bio‐Rad Laboratories, Hercules, CA) was used for detection. Membranes were reprobed with mouse anti‐actin antibody and anti‐mouse‐HRP antibody to normalize between samples. Protein abundance was quantified using Image Lab software (Bio‐Rad Laboratories, Hercules, CA).

After entrainment, seedlings were exposed to constant darkness, constant 60 μmol m⁻² s⁻¹ monochromatic blue or red light under LEDs (XtremeLUX, Santa Clara, CA), or 50–60 μmol m⁻² s⁻¹ white light at 22°C. Seedlings were moved at dawn (Time 0) and collected every 4 h from Times 0 to 48 (RVE4‐FLAG) or every 3 h from 3 h before dawn to Time 33 (RVE8‐HA). Samples were prepared and quantified as previously described (Shalit‐Kaneh et al., 2018 (link)). Total protein was analyzed by western blotting using mouse monoclonal anti‐FLAG M2‐HRP antibody (Sigma‐Aldrich, St. Louis, MO) for RVE4‐FLAG and rat monoclonal anti‐HA‐HRP antibody (Roche, Basel, Switzerland) for RVE8‐HA. Prometheus ProSignal Dura (Genesee Scientific, Rochester, NY) was used to generate peroxidase activity and a Chemidoc analyzer (Bio‐Rad Laboratories, Hercules, CA) was used for detection. Membranes were reprobed with mouse anti‐actin antibody and anti‐mouse‐HRP antibody to normalize between samples. Protein abundance was quantified using Image Lab software (Bio‐Rad Laboratories, Hercules, CA).