Phospho-TFEB (Ser142) Antibody - #AF3845

Fig. 2. Cd inhibits TFEB nuclear translocation (A) Venn diagram of DEG between the control and Cd-exposed group. (B) Heat map of DEGs of autophagy and lysosomal pathways in the control and Cd-exposed groups. (C) Renal tissues were collected to analyze the protein levels of TFEB. Representative immunoblot images were shown in upper panels and corresponding densitometric levels (mean ± SEM, n = 3) were shown in lower panels. (D) NRK-52E cells were treated with a range of Cd doses for 12 h to analyze the protein levels of TFEB. Representative immunoblot images were shown in upper panels and corresponding densitometric levels (mean ± SEM, n = 3) were shown in lower panels. (E) Renal tissues were collected to analyze the nuclear and cytoplasmic protein levels of TFEB. Representative immunoblot images were shown in upper panels and corresponding densitometric levels (mean ± SEM, n = 3) were shown in lower panels. (F) NRK-52E cells were treated with a range of Cd doses for 12 h to analyze the nuclear and cytoplasmic protein levels of TFEB. Representative immunoblot images were shown in upper panels and corresponding densitometric levels (mean ± SEM, n = 3) were shown in lower panels. (G) Detection of TFEB nuclear translocation in NRK-52E cells by IF staining method. Nuclear TFEB fluorescence intensity quantification in right panels. *p < 0.05 vs. control, **p < 0.01 vs. control.

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.)

Tat-Beclin1-induced autosis is regulated by Tfeb modulation in NRCMs. (A–C) NRCMs were treated with 5 µM Scrambled or Tat-Beclin 1 for 6 h and analyzed via Western blotting using anti-Tfeb, anti-pTfeb (Ser142) and anti-Gapdh antibodies (A) The relative ratios of Tfeb to Gapdh (B) and pTfeb to Tfeb (C) were quantified (mean values ± S.E., n = 6 for Tfeb, n = 3 for p-Tfeb; * p < 0.05, *** p < 0.001). (D,E) NRCMs were transduced with either Ad-lacZ or Ad-Tfeb for 24 h (D) or Ad-lacZ or Ad-shTfeb for 48 h (E) and then treated with 5 µM Scrambled or 0.5, 1 or 5 µM Tat-Beclin 1 for 3 h. Cell death was quantified via CellTiter-Blue assays (mean values ± S.D., n = 16; * p < 0.05, ** p < 0.01, not significant (n.s.)) (D,E). (F,G) Knockdown of Tfeb by Ad-shTfeb was confirmed via immunoblot analyses, using anti-Tfeb and anti-Gapdh antibodies (F). Knockdown of Atg7 by Ad-shAtg7 was confirmed via immunoblot analyses, using anti-Atg7 and anti-Gapdh antibodies (G). (H) NRCMs were transduced with either Ad-lacZ or Ad-Tfeb with or without Ad-shAtg7 as indicated for 48 h and then treated with 5 µM Scrambled or Tat-Beclin 1 for 3 h. Cell death was quantified via CellTiter-Blue assays (mean values ± S.D., n = 16; * p < 0.05, ** p < 0.01, not significant (n.s.)).