Phospho-DRP1 (Ser637) Antibody - #DF2980

Fig. 2: Pre-NRC starvation promotes mitochondrial tabulation. A The percentage of fragmented mitochondria in PG cells from 72 h AEL to WPP. From left to right, n = 30, 16, 27, 17, 24, and 40, respectively. B Mitochondrial morphology (green) was assessed in PG cells. Magnified images of the highlighted areas (red dotted box) are shown. Right: the percentage of fragmented mitochondria. Nucleus: DAPI (cyan). From left to right, n = 30, 23, 24, and 17, respectively. C–F Representative images of PTTH (C), p-ErK, green, 108 h: n = 24, pre-NRC ST: n = 27), insulin (D, E InR-SPARK and AKT-SPARK, green), and TORC1 signalings (F, p-S6K, green, 108 h: n = 9, pre-NRC ST: n = 8) in PG cells of control and pre-NRC ST larvae. G, H Representative images of mitochondrial morphology (green) in PG cells (168 h AEL) with inhibition of PTTH, insulin, TORC1 signalings G and mitochondria-associated genes H. Magnified images of the highlighted areas (red dotted box) are shown. G From left to right, n = 7, 7, 7, 6, 6, 13, 9, and 7, respectively. H From left to right, n = 32, 19, 19, 20, 12, 16, 17, and 19, respectively. I, J Images showing Marf (I Marf-EGFP, green) and p-Drp1 (J p-Drp1 Ser637 antibody, green) staining in PG cells of control and pre-NRC ST larvae at 108 h AEL. Mitochondria: mito-mCherry (red). Nucleus: DAPI (cyan). From left to right, n = 26, 15, 30, and 22, respectively in (I, J). K, L Images showing ATP-SPARK puncta (green) in PG cells of control and pre-NRC ST or Marf-expressing larvae at 108 h AEL. M, N Representative images of mitochondria morphology (M, green, From left to right, n = 20, 27, 24, and 19, respectively) and ATP-SPARK puncta (N, green) in PG cells (144 h AEL) with PG-specific expression of RasV12, Dp110CA, or Rheb. Magnified images of the highlighted areas (red dotted box) are shown. O, P Images showing p-AMPK levels (p-AMPK, green, 108 h: n = 17, pre-NRC ST: n = 19) O and AMPK-SPARK puncta (green) P in PG cells of controls and pre-NRC ST larvae at 108 h AEL. All Data are presented as mean ± SD. Different lowercase letters represent statistical significance by one-way ANOVA. The genotypes and statistical data are provided in Supplementary Data 1.

Figure 4 LFHP-1c targets PGAM5 to facilitate nuclear translocation of NRF2 for endothelial protection in stroke. (A) The expression of PGAM5, p-DRP1 (Ser637), NRF2 and HO-1 were detected at indicated times after LFHP-1c treatment in rBMECs by immunoblotting, n=3 per group. (B) and (C) IP/Western blotting results reveal that LFHP-1c treatment significantly reduced the interaction of PGAM5 with NRF2, n=3 per group. (D) LFHP-1c facilitates nuclear translocation of NRF2 in rBMECs, n=3 per group. (E) Silencing efficiency of siRNA against PGAM5 in rBMECs was verified by immunoblotting, n=3 per group. (F) and (G) LFHP-1c treatment or knockdown of Pgam5 facilitates nuclear translocation of NRF2 in rBMECs, and LFHP-1c treatment after si-PGAM5 did not affect the nuclear translocation of NRF2, n=3 per group. (H) Fluorescence staining of NRF2 shows that LFHP-1c treatment or knockdown of Pgam5 promoted NRF2 nuclear translocation, n=3 per group. The scale bar represents 20 μm. (I) and (J) The protein expressions of PGAM5, p-DRP1 (Ser637), NRF2 and HO-1 in rat brain microvessels were detected at 72 h after tMCAO onsetand representative blots are shown here, n=4 per group. (K) The mRNA expression of Ho-1 and Nqo1 was measured by RT-PCR, n=4–5 per group. Results are expressed as mean±SEM. For Fig. 4C, *** P<0.001 versus IgG plus DMSO group, ## P<0.01 versus PGAM5 plus LFHP-1c (2 μmol/L) group. For Fig. 4D, * P<0.05, *** P<0.001 versus ‘0’ time point group. For Fig. 4E, *** P<0.001 versus scramble group. For Fig. 4G, *** P<0.001 versus scramble plus DMSO group. For Fig. 4J and K, * P<0.05, ** P<0.01, *** P<0.001 versus Sham group; # P<0.05, ## P<0.01, ### P<0.001 versus Vehicle group.

FIGURE 8 | PGC-1α might be important in mediating the effects of PF on TNF-α-treated C2C12 cells.(K) Representative western blots using antibodies against p-DRP1 (Ser637), DRP1, MFN1, MFN2, OPA1, and GAPDH.

Fig. 8 PD150606 inhibited mitochondrial fragmentation by modulating Drp-1 phosphorylation. a–c The changes of p-Drp- 1(Ser637) and calcineurin A in NRCMs infected with CVB3 with or without PD150606. PD150606 treatment inhibited Drp-1 dephosphorylated at the Ser637 site (b) and calcineu- rin activation (c) after CVB3 infection. d–h The changes of p-Drp-1(Ser637) and apoptosis-related proteins in NRCMs infected with CVB3 with or without FK506 (an inhibitor of calcineurin activation). Dephosphorylation of Drp-1 (e) was activated by calcineurin activation and contributes to apoptosis (f–h) in CVB3-infected NRCMs. *P < 0.05 vs. Con; #P < 0.05 vs. virus group

FIGURE 5 (A) The molecular structure of AB4. (B–D) Molecular docking of AB4 with GSK‐3β. The modelled 3D structure of GSK‐3β docked with AB4 (B). The enlarged view of binding site in box (C). The interaction bonds of GSK‐3β with AB4 (D). Bonds showed as yellow dotted lines, and bond lengths were presented as numbers. (E) The titration between AB4 and GSK‐3β. The top panel presents typical calorimetric titration of AB4 with GSK‐3β at 25°C. The bottom panel shows the plots of the heat evolved (kcal) per mol of AB4 added corrected for the heat of with GSK‐3β, against the molar ratio of AB4 to GSK‐3β. Data solid squares were fitted to a single set of the identical sites model, and the solid line represented the best fit. (F) Representative immunofluorescence staining images of GSK‐3β and Drp1 in the spinal dorsal horn of the control, CIA and CIA + AB4 groups. Scale bar = 20 μm. (G) Quantitative analysis of the fluorescence intensity of GSK‐3β and Drp1. (H, I) Western blot analysis and quantitative grey value analysis of pGSK‐3β‐Tyr216, GSK‐3β, pDrp1‐Ser616, pDrp1‐Ser637 and Drp1 level in the spinal cord of the control, CIA and CIA + AB4 groups. Data are presented as mean ± SD (n = 5). *p