primary antibodies targeting lc3  (Cell Signaling Technology Inc)

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: Primary antibodies LC3 (1:100, Cell Signaling Technology, #12741), Keap1(1:100, Zen-bio, R26935 ) and Nrf2 (1:100, Zen-Bio, 380773) were incubated overnight at 4 °C.

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

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: TiO₂ NZs impair fetal growth and induce placental energy deficiency and autophagy. ( A ) Morphology of TiO₂ NZs and bulk TiO₂ (b-TiO₂) observed by TEM.Scale bars: 100 nm for TiO₂ NZs and 1 μm for b-TiO₂. ( B ) Key characteristics of the TiO₂ NZs and b-TiO₂ used in this study. ( C ) A schematic diagram illustrating the animal study design and key time points. ( D ) Maternal weight during pregnancy in the control ( n = 8), TiO₂ NZs ( n = 8), and b-TiO₂ ( n = 8) groups. ( E ) Total fetal weight, average fetal weight, and average fetal length in the control ( n = 122), TiO₂ NZs ( n = 113), and b-TiO₂ ( n = 118) groups. ( F ) The average fetal number in the control group ( n = 8), TiO₂ NZs exposure group ( n = 8), and b-TiO₂exposuregroup ( n = 8). The data are presented as mean ± SD. ( G ) Representative images of fetuses from the control, TiO₂ NZs, and b-TiO₂ treatment groups. ( H, I ) Placental weight and ATP content in the control ( n = 5), TiO₂ NZs ( n = 5), and b-TiO₂ ( n = 5) groups. ( J ) Western blot analysis of LC3-I and LC3-II expression levels in placental tissues from the control group ( n = 3) and the TiO₂ NZs group ( n = 3). GAPDH served as a loading control.The ratio of LC3-II to LC3-I was quantified by densitometry.Data are presented as mean ± SD. One-way ANOVA was used for ( E , F , H and I ). An unpaired two-tailed t-test was used for ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control

Article Snippet: Autophagosomes were labeled by sequential incubation with an anti-LC3 primary antibody (4108, Cell Signaling Technology, Danvers, MA, USA)overnight at 4 °C and a CY3-conjugated goat anti-rabbit secondary antibody (Abcam, UK, ab6939).

Techniques: Control, Western Blot, Expressing, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Phenformin and Compound C rewire AMPK/mTOR pathway in HTR cells post-TiO₂ exposure. ( A, B ) Western blot analysis of Akt, AMPK, mTOR, and their phosphorylated forms after cells were treated with 100 µg/mL TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector (pcDNA3.1-AMPK), or TiO₂ NZs + AMPK overexpression vector + Compound C/phenformin for 24 h. ( C ) Protein levels of AKT, p-AKT, AMPK, p-AMPK, mTOR, p-mTOR, and LC3 in the control group, TiO₂ NZs exposure group, TiO₂ NZs + phenformin and TiO₂ NZs + Compound C groups, as determined by western blotting. GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands. Quantitative densitometric analysis corresponding to the western blots in ( A – C ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times.Data were collected from three independent experiments and presented as mean ± SD. An unpaired two-tailed t -test was used for (C left panel). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( A , B , C right panel). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 TiO₂ NZs + AMPK-vector + Compound C vs. TiO₂ NZs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 TiO₂ NZs + AMPK-vector vs. TiO₂ NZs group; † P < 0.05, †† P < 0.01, ††† P < 0.001 TiO₂ NZs + AMPK-vector + Compound C vs. TiO₂ NZs + AMPK-vector group (A-B). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ### P < 0.001 TiO₂ NZs + Compound C vs. TiO₂ NZs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 TiO₂ NZs + phenformin vs. TiO₂ NZs group ( C )

Article Snippet: Autophagosomes were labeled by sequential incubation with an anti-LC3 primary antibody (4108, Cell Signaling Technology, Danvers, MA, USA)overnight at 4 °C and a CY3-conjugated goat anti-rabbit secondary antibody (Abcam, UK, ab6939).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Control, Software, Expressing, Two Tailed Test, Comparison

Journal: Oncology Letters

Article Title: ERK-driven autophagy enhances synergy of eribulin and cisplatin in triple-negative breast cancer

doi: 10.3892/ol.2025.15274

Figure Lengend Snippet: Effects of eribulin and cisplatin on autophagy in MDA-MB-231 cells. (A) Western blot analysis of autophagy-related proteins LC3-I/II and p62. β-actin was used as a loading control. The levels of autophagy-associated proteins (B) LC3-I/II and (C) p62 were semi-quantified. Β-Actin was used as a loading control. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.005. (D) Autophagy was measured using an autophagy assay kit. Cells were treated with the indicated compounds (60 µM), labeled and analyzed by fluorescence microscopy. Green staining indicates autophagosomes and blue represents DAPI nuclear staining. Scale bar, 100 µm. (E) Quantification of the mean fluorescence intensity using ImageJ software. Data are presented as the mean ± SD. **P<0.005.

Article Snippet: Primary antibodies against LC3-I/II (cat. no. 4108), phospho-ERK1/2 (Thr202/Tyr204; cat. no. 9101), and β-actin (cat. no. 4967) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Control, Labeling, Fluorescence, Microscopy, Staining, Software

Journal: Oncology Letters

Article Title: ERK-driven autophagy enhances synergy of eribulin and cisplatin in triple-negative breast cancer

doi: 10.3892/ol.2025.15274

Figure Lengend Snippet: Effects of ERK inhibition on eribulin and cisplatin-induced changes in cell viability, colony formation, apoptosis and autophagy. (A) Following pre-incubation with PD98059 before eribulin and cisplatin treatment, cell viability was determined using the Cell Counting Kit-8 assay. Each assay was performed in triplicate. Data are presented as the mean ± SD. **P<0.005. (B) The apoptotic cell population was evaluated by flow cytometry following double staining with annexin V and PI. (C) Quantitative results of annexin-PI flow cytometry. The percentage of apoptotic cells is presented as the mean ± SD. **P<0.005. (D) Western blot analysis of autophagy-related proteins. Levels of autophagy-associated proteins (E) LC3-I/II and (F) p62 were semi-quantified. β-Actin was used as a loading control. Data are presented as the mean ± SD of three independent experiments. **P<0.005. (G) Autophagy was measured using an autophagy assay kit (ab139484; Abcam). Cells were treated with the indicated compounds (60 µM eribulin, 60 µM cisplatin and 25 µM PD98059), labeled and analyzed using fluorescence microscopy. Green staining indicates autophagosomes and blue represents DAPI nuclear staining. Scale bar, 100 µm. (H) Quantification of the mean fluorescence intensity using ImageJ software. Data are presented as the mean ± SD. **P<0.005 vs. eribulin + cisplatin group.

Article Snippet: Primary antibodies against LC3-I/II (cat. no. 4108), phospho-ERK1/2 (Thr202/Tyr204; cat. no. 9101), and β-actin (cat. no. 4967) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Incubation, Cell Counting, Flow Cytometry, Double Staining, Western Blot, Control, Labeling, Fluorescence, Microscopy, Staining, Software