anti rabbit p62  (Proteintech)

Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: TIGAR activated nuclear factor erythroid-2 related factor (Nrf2) to reduce dexamethasone (Dex)-induced oxidative stress through inducing autophagy. Bone marrow mesenchymal stem cells (BMSCs) were transfected with TIGAR overexpression plasmid and treated with Dex. (A, B) Immunofluorescence staining of Nrf2 of BMSCs and the quantification of the integrated optical density (IOD) per field. Scale bars, 50 μm. (C, D) Western blot analysis and quantification of Nrf2 expression in extracted nuclear proteins. (E, F) ROS level under Dex treatment in BMSCs with or without administration of 10 nM Nrf2 inhibitor (ML385) after transfecting with TIGAR overexpression plasmid, and the quantification of IOD per filed. Scale bars, 100 μm. BMSCs were treated with Dex and chloroquine (CQ) (20 μM) after transfecting with TIGAR overexpression plasmid. (G–I) Western blot analysis and quantification of p62 and LC3-II expression under different treatments. (J, K) Representative images of mCherry-GFP-LC3 puncta and number of autophagosomes (yellow) (analyzed by Pearson's correlation). Scale bars, 50 μm. (L–N) Western blot analysis and quantification of Nrf2 and kelch-associated protein 1 (Keap1) expression under different treatments. (O, P) The immunofluorescence staining of Nrf2 in BMSCs and the quantification of the IOD per field. Scale bars, 50 μm. (Q, R) Representative immunofluorescence images of LC3 and Keap1. Pearson's correlation of co-localization is shown in the bar graph format from the three independent experiments analyzed. Scale bars, 50 μm. (S, T) Representative images of ROS and the quantification of the IOD per field. Scale bars, 100 μm. Data are shown as mean ± SEM. n = 3, biologically independent samples. Two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used to assess statistical significance. ∗ p < 0.05, ∗∗ p < 0.01. NC, negative control. OE, TIGAR overexpression plasmid.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Transfection, Over Expression, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Expressing, Negative Control

Journal: Renal Failure

Article Title: Aucubin ameliorates diabetic kidney disease by restoring hGENCs autophagy through promoting phosphorylation of ATG4B protein

doi: 10.1080/0886022X.2025.2605756

Figure Lengend Snippet: Protective effect of aucubin on high glucose-induced hGENCs injury by promoting ATG4B protein phosphorylation and increasing autophagy. (A) Electrophoretic map of cells in each group. 1, 2, 3, 4, and 5 represent Con group, HG group, HG-AU-L group, HG-AU-M group, and HG-AU-H group, respectively. (B) Mass spectrogram of protein identification. Venn diagram of (C) phosphorylated proteins, (D) phosphorylated protein sites and (E) protein identification, AU1, AU2 and AU3 represent HG-AU-L group, HG-AU-M group, and HG-AU-H group, respectively. (F) Representative images of expression levels of p-ATG4B and ATG4B detected by WB. (G) Representative images of the expression levels of autophagy protein LC3, p62, ATG5, and ATG7 in each group detected by WB, and the statistical analysis result of the proteins expression levels. *p < .05; ** p < .01. AU: aucubin; HG: high glucose.

Article Snippet: Proteins were transferred to polyvinylidene difluoride membranes (PVDF) and then incubated with specific primary antibodies against ATG4B, p-ATG4B(Ser383), ATG5, ATG7 (1:1,000, CST); Bcl-2, Bax (1:500, Immunoway); p62, cleaved caspase3 (1:500, Immunoway); LC3 (1:1,000, Abcam); CD31, Vimentin (1:2,000, Proteintech); nephrin (1:1,000, Affinity) and α-SMA, GAPDH(1:1,000, Proteintech), and secondary antibodies conjugated to HRP (Proteintech).

Techniques: Phospho-proteomics, Expressing

Journal: Renal Failure

Article Title: Aucubin ameliorates diabetic kidney disease by restoring hGENCs autophagy through promoting phosphorylation of ATG4B protein

doi: 10.1080/0886022X.2025.2605756

Figure Lengend Snippet: Protective effect of aucubin on DKD mice by promoting ATG4B protein phosphorylation and increasing autophagy. The expression levels of p-ATG4B proteins, ATG4B proteins, autophagy protein (LC3, p62, ATG5, and ATG7), apoptosis-related proteins (Bax, cleaved caspase3, and Bcl-2), EndMT marker proteins (CD31 and α-SMA), and nephrin protein in each group were detected by WB and the statistical analysis result of the proteins expression levels. *p < .05; ** p < .01. DKD: diabetic kidney disease; AU: aucubin.

Article Snippet: Proteins were transferred to polyvinylidene difluoride membranes (PVDF) and then incubated with specific primary antibodies against ATG4B, p-ATG4B(Ser383), ATG5, ATG7 (1:1,000, CST); Bcl-2, Bax (1:500, Immunoway); p62, cleaved caspase3 (1:500, Immunoway); LC3 (1:1,000, Abcam); CD31, Vimentin (1:2,000, Proteintech); nephrin (1:1,000, Affinity) and α-SMA, GAPDH(1:1,000, Proteintech), and secondary antibodies conjugated to HRP (Proteintech).

Techniques: Phospho-proteomics, Expressing, Marker