Journal: Nature Communications

Article Title: Phytochrome B and phytochrome-interacting-factor4 modulate tree seasonal growth in cold environments

doi: 10.1038/s41467-025-63391-5

Figure Lengend Snippet: a Western blot of PIF4a-HA fusion protein in PIF4a-HA overexpressing plants in response to red light at 21 °C and 15 °C using an anti-HA antibody to detect PIF4a-HA and an anti-RPN6 antibody as loading controls. 4-week-old plants were kept in the dark at 21 °C for 24 h and then transferred into two cabinets with 10 μmol m −2 s −1 red light at either 21 °C or 15 °C. Leave samples were taken at 0, 10, 30, and 60 min of red light treatment at two temperatures. The bottom panel shows the quantification of PIF4a-HA fusion protein by western blot. Data are represented as mean ± SEM, n = 3. The asterisks represent significance levels (* p < 0.05) based on Fisher’s LSD test (two-sided) without multiple testing correction following ANOVA. b TUBEs (tandem ubiquitin-binding entities) assays of ubiquitinated proteins in the samples from ( a ) after 30 and 60 min of red light treatment. Total ubiquitinated proteins from samples were immunoprecipitated with agarose-TUBE2, then analyzed by western blotting with anti-HA antibodies for detection of PIF4-HA and anti-ubiquitin antibodies as loading controls. Control agarose that was not TUBEs-conjugated was used as a negative control. Anti-RPN6 antibodies were used as loading controls for input samples. c Co-immunoprecipitation assays of PHYB2 and PIF4a interaction in 35S::PIF4a-HA-1/35S::PHYB2-GFP trees at 21 °C and 15 °C. Co-immunoprecipitated PIF4a-HA was detected by western blotting using anti-HA antibodies, after immunoprecipitation of PHYB2-GFP by GFP-trap beads. Precipitate probed with anti-GFP antibodies and input samples are shown as controls. d TUBEs assays of ubiquitinated proteins in 35S::PIF4a-HA-1 and 35S::PIF4a-HA-1/35S::PHYB2-GFP trees at two temperatures. Leaf samples were taken at ZT8 of 6-week-old LD grown trees. One short-time exposed image and one long-time exposed image are shown from the same blot. See also Supplementary Fig. . Source data are provided as a file.

Article Snippet: The agarose beads were washed with extraction buffer four times and eluted with 2x laemli loading buffer, then subjected to Western blot analysis with the 16B12 anti-HA-POD antibodies (Roche) for detection of PIF4-HA and anti-ubiquitin antibodies (sc-8017, Santa Cruz Biotechnology) for detection of ubiquitinated protein.

Techniques: Western Blot, Ubiquitin Proteomics, Binding Assay, Immunoprecipitation, Control, Negative Control