Phospho-ULK1 (Ser757)[Ser758] Antibody - #AF4387

Fig. 7. rhFGF21 promotes autophagic degradation of HIF-1α. (A, B) Representative immunofluorescence and quantification of colocalization of HIF-1α and Lamp1 in BMDMs from indicated group (n = 5). DAPI (blue), HIF-1α (red), and Lamp1 (green). Scale bars: 20 μm. (C) The expression of HIF-1α in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 5). Bafilomycin A1 was added for the last 6 h. (D) BMDMs were treated as indicated, and the cell lysates were subjected to immunoprecipitation with HIF-1α or p62 antibody, followed by immunoblotting with the HIF-1α or p62 antibody. MG132 (10 μM) and Bafilomycin A1 (100 nM) were added for the last 6 h. (E) The expression of HIF-1α in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 5). BMDMs were transfected with Si-scramle (20 nM) and Si-sqstm1 (20 nM) for 48 h, then BMDMs were treated with single LPS or LPS + rhFGF21 for 12 h (n = 4). (F) The expression of p-ULK1 (Ser757) and p-p62 (Ser405) in lysates of BMDMs in indicated groups, and normalized to ULK1 and p62 (n = 4). (G, H) Representative immunofluorescence and quantification of HIF-1α-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), HIF-1α (red), and F4/80 (green). White triangles indicate HIF-1α-F4/80 double positive macrophages. Scale bars: 50 μm. (I) The expression of HIF-1α, LC3 and p62 in Kupffer cells isolated from liver of mice in indicated groups (n = 5). β-actin was used as the standard for verifying equivalent loading. (J) Real-time PCR analysis for hk2, pkm2, and ldha in liver from mice in indicated groups. Data are presented as mean ± SEM of each group. And one-way ANOVA test is using to assess the statistical differences in multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

FIGURE S1 | Evaluation of autophagic activity in a SCI rat model treated with exosomes. (a, b) Representative images of western blots used to determine the expression levels of AMPK, p-AMPK, S6K, p-S6K, ULK1, p-ULK1, and p62 and semiquantification of the data. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group by t test or ANOVA. #P < 0.05 compared with the Exos group by t test. n = 3 for each group.

Figure 2 The effects of HICA on the intracellular signaling pathways. A typical image for a capillary immunoassay is shown (A). The phosphorylation levels of (B) p70S6K and 4E-BP1; (C) AMPK, ACC, and ULK1; (D) ERK1/2; (E) p38MAPK; and (F) eEF2 are shown. The phosphorylation is normalized to the total protein expression. The β-tubulin content in the lysate was measured as a loading control (G). The time course of these experiments is shown in the upper region. DM: differentiation medium, DMEM: Dulbecco’s modified Eagle’s medium, and w/o AA: without amino acids. Data are displayed as the means ± SD, and n = 4 for each group in all bar graphs. * p < 0.05 and ** p < 0.01 vs. the vehicle-treated group.

Fig. 4. ICA regulated the AMPK/mTOR/ULK1 signaling pathway. A–C Mode of ICA binding with AMPK, mTOR, and ULK1. The chemical structure in orange indicates ICA, and the purple structure indicates the binding site of ICA, AMPK, mTOR, and ULK1. D, E The effect of ICA on the expression of AMPK, ULK1, and mTOR and their phosphorylation in MDA-MB-468 cells; F, G in 4T1 cells. Bars represent the means ± SD of at least three independent experiments; ns, not significant; *P 

Figure 2 Taurine treatment decreases mTOR activity and inhibits phosphorylation of ULK1 and ATG13 in MAC-T cells. (A–D) Immunoblots of total protein from MAC-T cells treated with 70 mmol·L−1 taurine for different durations. Quantification of the ratio between the total and phosphorylated (p–) proteins, as determined by densitometric scanning of immunoblots (n = 3). Data are presented as mean ± SEM. (E–G). Cells were incubated with 50 nmol·L−1 Baf-A1 for 1 h, and then treated with 70 mmol·L−1 taurine for 4 h or 100 nmol·L−1 Rapa for 2 h or both; all cells were then probed for the total and phosphorylated (p–) proteins shown. Data are presented as mean ± SEM. *P < 0.05 (significantly different) between the indicated groups; #P < 0.05 (significantly different) between the corresponding negative control groups.