Phospho-SQSTM1/p62 (Ser403) Antibody - #DF2985

Fig. 3. LPS impaired autophagic flux in macrophages. (A, B) Representative immunofluorescence and quantification of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. Scale bars: 50 μm. (C, D) Representative immunofluorescence and quantification of LC3 puncta in F4/80-positive macrophages in liver from mice in indicated group. DAPI (blue), LC3β (red), and F4/80 (green) (n = 5). Scale bars: 50 μm. (E, F) Representative immunofluorescence and quantification of p62-F4/80 double positive macrophages in liver from mice in indicated groups. DAPI (blue), p62 (red), and F4/80 (green) (n = 5). White triangles indicate p62-F4/80 double positive macrophages. Scale bars: 50 μm. (G) The expression of LC3 and p62 in lysates of BMDMs in “control-group” and “LPS-group”, and normalized to β-actin (n = 4). (H) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). And Bafilomycin A1 was added for last 6 h. (I, J) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-mCherry-LC3B (n = 10). Scale bars: 20 μm. (K) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). 3-MA was given as pretreatment for 12 h before LPS administration. (L, M) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-LC3B in indicated groups (n = 8). Scale bars: 10 μm. (N) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). U0126, SP600125 and SB203580 were pretreated to BMDMs for 12 h. (O, P) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-LC3B in indicated groups (n = 8). Scale bars: 20 μm. (Q, R) Representative confocal images and quantification of pHLys Red in indicated groups (n = 5). Scale bars: 50 μm. (J) Real-time PCR analysis for lamp1, ctsa, ctsb, and ctsf from BMDMs in indicated group. All values are presented as mean ± SEM. Statistical significance was measured using the unpaired 2-tailed Student t test for two experimental groups and one way ANOVA test for 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.)

Supplementary Figure. S6| HEK293 cells were transfected with GFP or GFP-CRBN for 48 h and the cell lysates were subjected to immunoblotting analysis using the indicated antibodies.

Fig. 3. Sestrin2 overexpression activates the Nrf2 signaling pathway and increases VEGF expression. (a, b) Representative WB images of sestrin2, p-p62, p62, total Nrf2, HO-1, VEGF and nuclear Nrf2 expression. (c-h) Quantitative WB analysis of sestrin2, p-p62, p62, total Nrf2, HO-1, VEGF and nuclear Nrf2 expression. The data are presented as the mean ± SEM, n = 9 animals/group. NS, not significant, *, p < 0.05, **, p < 0.01, ***, p < 0.001 versus the PTI group.

Figure 6: |ATL-III ameliorated TNF-α-induced apoptosis in C2C12 myoblasts through the PI3K/AKT/mTOR pathway. C2C12 myoblasts were incubated with ATL-III in the presence or absence of TNF-α (20 ng/ml) for 24 h. After treatment and protein quantification, the expression levels of p-PI3K, p-AKT (Ser473), p-mTOR, LC3, and P62 were tested by western blotting. GAPDH was used as a loading control. The signaling pathway was explored again after overexpression of Nox2 (a).