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. 2023 Dec 21;120(52):e2318274120. doi: 10.1073/pnas.2318274120

Fig. 7.

Fig. 7.

PER, BMAL1, and CRY1 microbodies and their interactions revealed by STED microscopy. (A and B) STED microscopy images of PER2 and BMAL1/CRY1 puncta in the PER2-EGFP KI cells at 10 h after synchronization. PER2, BMAL1, and CRY1 were stained by anti-GFP (mouse), anti-BMAL1 antibody (rabbit), and anti-CRY1 antibody, respectively. (Scale bar: 5 μm.) (C and D) Percentages of PER2 and BMAL1/CRY1 bodies that colocalize at 10 h after synchronization. Distance threshold was set <100 nm. Data are presented as mean ± SEM (n = 8 cells). (E) Comparison of PER body sizes estimated by conventional anti-GFP antibody and anti-GFP nanobody. n = 5 cells. (F and G) Immunodepletion assay of CRY1 or BMAL1 in extracts prepared from the indicated double KI U2OS cells 10 h after synchronization (F) or from mice liver tissue (ZT18) (G). After immunodepletion, western blot analyses were performed using the indicated antibodies. (H) Representative immunofluorescence imaging results in the nucleus using mice SCN tissues harvested at the indicated ZT time points using a rabbit polyclonal PER2 and a mouse CRY1 antibody, respectively. Cells from the core SCN region were selected. (I) Quantification of the nuclear PER2/CRY1 foci numbers in the SCN cells at the indicated time points.