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.

Fig. 8. Western blotting results for AMPK, p-AMPK, TSC1, mTOR, p-mTOR, ULK1, p-ULK1.

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.