phospho ulk1 ser555  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Activation of the protective arm of renin-angiotensin system enhances mitochondrial turnover improving respiration and decreasing integrated stress response in a human Complex III deficiency model

doi: 10.64898/2026.03.20.711686

Figure Lengend Snippet: (A) Representative blots of phosphorylated and total AMPK in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 10 and 30 minutes. Vinculin was used as loading control. ( B ) Average expression of p-AMPK by total AMPK levels in Ctrl vs CIII-deficient, and ( C ) CIII-deficient untreated and treated with CAP-1902. Statistical analysis was performed using a t-test and one-way ANOVA, respectively. (D) Representative blots of phosphorylated and total AMPK in CIII-deficient untreated and treated with 1 μM A779 for 1h, plus and minus 5 nM CAP-1902 for the last 30 minutes. ( E ) Average expression of p-AMPK by total AMPK levels. Statistical analysis was performed by one-way ANOVA. (F) Representative blots of phosphorylated (S638) ULK1 and total ULK1 in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. ACTIN was used as loading control. (G) Average expression of p-ULK1 by total ULK1 levels in Ctrl vs CIII-deficient untreated and ( H) CIII-deficient untreated and treated with CAP-1902. Statistical analysis was performed by unpaired t-test. (I) Representative blots of ULK1 in mitochondria-enriched fractions (M) vs the corresponding supernatant (S) in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. Citrate synthase is included as a control for mitochondrial enrichment, and total protein stain as loading control. (J) Quantification of the expression of ULK1/total protein in each fraction. Statistical analysis was performed by two-way ANOVA. (K) Representative blots of phosphorylated (S17) FUNDC1 and total FUNDC1 in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. ACTIN was used as loading control. (L) Average expression of p-FUNDC1 by total FUNDC1 levels in Ctrl vs CIII-deficient untreated and (M) CIII-deficient untreated and treated with CAP-1902. Statistical analysis was performed by unpaired t-test. ( N ) Number of mitolysosomes in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. Quantification represents the average number of LC3-II puncta touching mitochondria (mitophagosomes) per cell. Statistical analysis was performed by two-way ANOVA. ( O ) Confocal micrographs of Ctrl, CIII, and CIII cells treated with 5 nM CAP-1902 ± bafilomycin for 16 h, labeled with LC3B (autophagosome – red), GRP75 (mitochondria - green), and DAPI (nuclei - blue). Scale bars: 100 μm and 5 μm in the zoomed image. In all cases, data show mean ± SEM. Dots in graphs represent independent biological replicates.

Article Snippet: The primary antibodies used were: anti-human OXPHOS cocktail (Abcam, ab11041); anti-PROHIBITIN (Abcam, ab28172); anti-CITRATE SYNTHASE (Abcam, ab96600); anti-MIC60 (Abcam, ab137057); anti-TOMM20 (Sigma-Aldrich, WH0009804M1); anti-phospho AMPK (T172) (Cell Signaling, 2535); anti-AMPK (Cell Signaling, 2532); anti-phospho FUNDC1 (S17) (Invitrogen, PA5-114576); anti-FUNDC1 (Aviva Systems Biology, ARP53280_P050); anti-phospho ULK1 (S638) (Cell Signaling, 14205); anti-ULK1 (Cell Signaling, 8054); anti-LAMP1 (Cell Signaling, 51774); anti-MasR (Santa Cruz Biotechnology, sc-390453); anti-GST-Tag (Cell Signaling, 2624); anti-LC3B (Cell Signaling, 3868); anti-CHOP (Cell Signaling, 2895); anti-LAMIN A/C (Santa Cruz Bio, sc-20681); anti-β3 TUBULIN (Cell Signaling, 5568); anti-VINCULIN (Sigma-Aldrich, V9131); anti-ACTIN (Sigma-Aldrich, MAB1501), anti-TOMM20 Alexa Fluor 488 (Abcam, ab205486); anti-GRP75 (NeuroMab, 75-127); anti-ATF4 (Cell Signaling, 11815); anti-PGC1α (Invitrogen, PA5-72948); anti-SDHA (Invitrogen, 459200); anti-phospho ACC (S79) (Abcam, ab68191); anti-ACC (Abcam, ab45174).

Techniques: Control, Expressing, Staining, Labeling