anti lc3a b  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Excessive ER-mitochondria coupling: A DRP1-driven mechanism underlying mitochondrial dysfunction and impaired autophagy in stress-induced depression-like behavior in mice

doi: 10.1016/j.redox.2026.104121

Figure Lengend Snippet: CSDS upregulates Drp1 expression in hippocampal neurons. A) Differential protein volcano map of CSDS group and Control group (n = 3 samples per group; each sample pooled from 6 hippocampi). Red: significantly upregulated (fold change >1.5); blue: significantly downregulated (fold change >1.5); gray: non-significant. B) CO-IP analysis of Drp1 protein and its ubiquitination level in hippocampus of three groups of mice. One-way ANOVA with Tukey's multiple comparisons test (Treatment, F (2, 15) = 162.8, P < 0.0001; Treatment, F (2, 15) = 12.10, P = 0.0007). Data are expressed as mean ± SEM (n = 6 mice per group). ∗∗ p < 0.01, ∗∗∗ p < 0.001 C) Representative images of Drp1 (green), MAP2 (neuron, red) and DAPI (nucleus, blue) in CA1 of two groups (left) and Pearson correlation coefficient was quantitatively analyzed (right, n = 5 mice brain slices). Scale bar: 50 μm. D) Experimental timeline of viral injection and CSDS modeling; Representative confocal Z-stack 3D reconstructed images of dendritic spines in CA1 (left) and dendritic spines was quantitatively analyzed (right, n = 5 mice brain slices). Scale bar: 2 μm. Student's t -test (P = 0.0041; P = 0.0006). Data are expressed as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001. E) Representative images of Drp1 (green), VGLUT2 (Neuron, red) and DAPI (nucleus, blue) in hippocampal CA1 region of two groups (left) and the percentage of co-localized fluorescence was quantified (right). Scale bar: 50 μm. Two-way ANOVA with Tukey's multiple comparisons test (Interaction, F (2, 24) = 2.671, P = 0.0897). The data were expressed as mean ± SEM (n = 5 mice brain slices). ns, p > 0.05.

Article Snippet: Multiplex staining was performed using TSA-based immunofluorescence with primary antibodies: Drp1 (CST 12741S, 1:100), MAP2 (ab32454, 1:1000), GFAP (CST 12389, 1:400), IBA1 (ab178846, 1:1000), Vglut1 (CST #27181, 1:800), Vglut2 (ab216463, 1:250), OLIG2 (CST #65915, 1:400), and LAMP1 (CST 99437S, 1:100).

Techniques: Expressing, Control, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Injection, Fluorescence

Journal: Redox Biology

Article Title: Excessive ER-mitochondria coupling: A DRP1-driven mechanism underlying mitochondrial dysfunction and impaired autophagy in stress-induced depression-like behavior in mice

doi: 10.1016/j.redox.2026.104121

Figure Lengend Snippet: Drp1 mediates synaptic dysfunction in hippocampal CA1 neurons following CSDS. A) Experimental timeline of viral-mediated Drp1 knockdown, CSDS, and behavioral tests. B) The representative Western blot images (left) and corresponding protein statistical maps (right) of Drp1 in the hippocampus of the two groups. Student t-test (P < 0.0001). The data were expressed as mean ± SEM (n = 6 mice in each group). ∗∗ p < 0.01. C) The representative trajectory heat map of the control group and CSDS mice in the SIT. Behavioral tests including SIT (Interaction F (1, 44) = 35.27, P < 0.0001; Interaction, F (3, 88) = 2.920, P = 0.0385), SPT (Interaction, F (1, 44) = 7.745, P = 0.0079) and FST (Interaction, F (1, 44) = 6.636, P = 0.0134) were performed in mice. Two-way ANOVA with Tukey's multiple comparisons test. Data are expressed as mean ± SEM (n = 12 mice in each group). ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. D) The representative traces, amplitude (Interaction, F (1, 16) = 0.1631, P = 0.6917) and frequency (Interaction, F (1, 16) = 4.449, P = 0.0510) statistical maps recorded by sEPSC and the frequency accumulation map of sEPSC in CA1 neurons. Two-way ANOVA with Tukey's multiple comparisons test. Data are expressed as mean ± SEM (n = 5 mice in each group). ns, p > 0.05, ∗ p < 0.05.

Article Snippet: Multiplex staining was performed using TSA-based immunofluorescence with primary antibodies: Drp1 (CST 12741S, 1:100), MAP2 (ab32454, 1:1000), GFAP (CST 12389, 1:400), IBA1 (ab178846, 1:1000), Vglut1 (CST #27181, 1:800), Vglut2 (ab216463, 1:250), OLIG2 (CST #65915, 1:400), and LAMP1 (CST 99437S, 1:100).

Techniques: Knockdown, Western Blot, Control

Journal: Redox Biology

Article Title: Excessive ER-mitochondria coupling: A DRP1-driven mechanism underlying mitochondrial dysfunction and impaired autophagy in stress-induced depression-like behavior in mice

doi: 10.1016/j.redox.2026.104121

Figure Lengend Snippet: Drp1 overexpression enhances stress susceptibility in mice. A) Experimental timeline of Drp1 overexpression, subthreshold social defeat stress (sub-SDS), and behavioral testing. B) Western blot analysis (top) and quantification (bottom) of Drp1 expression in the hippocampus of the two groups. Student t-test (P < 0.0001). The data were expressed as mean ± SEM (n = 6 mice in each group). ∗∗∗ p < 0.001. C) Representative SIT trajectory heatmaps. Behavioral tests including SIT (Interaction, F (1, 44) = 9.435, P = 0.0036; Interaction, F (3, 88) = 2.974, P = 0.0360), SPT (Interaction, F (1, 44) = 2.772, P = 0.1030) and FST (Interaction, F (1, 44) = 3.270, P = 0.0774) were performed in mice. Two-way ANOVA with Tukey's multiple comparisons test. Data are expressed as mean ± SEM (n = 12 mice in each group). ns, p > 0.05, ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: Multiplex staining was performed using TSA-based immunofluorescence with primary antibodies: Drp1 (CST 12741S, 1:100), MAP2 (ab32454, 1:1000), GFAP (CST 12389, 1:400), IBA1 (ab178846, 1:1000), Vglut1 (CST #27181, 1:800), Vglut2 (ab216463, 1:250), OLIG2 (CST #65915, 1:400), and LAMP1 (CST 99437S, 1:100).

Techniques: Over Expression, Western Blot, Expressing

Journal: Redox Biology

Article Title: Excessive ER-mitochondria coupling: A DRP1-driven mechanism underlying mitochondrial dysfunction and impaired autophagy in stress-induced depression-like behavior in mice

doi: 10.1016/j.redox.2026.104121

Figure Lengend Snippet: Drp1 knockdown rescues CSDS-induced MERC remodeling and autophagy impairment. A) Mitochondrial respiration parameters including basal respiration (Interaction, F (1, 20) = 9.360, P = 0.0062), ATP production (Interaction, F (1, 20) = 15.14, P = 0.0009) and maximum respiratory capacity (Interaction, F (1, 20) = 0.9504, P = 0.3413). Two-way ANOVA with Tukey's multiple comparisons test. Data are expressed as mean ± SEM (n = 6 mice in each group). ns, p > 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. B) Representative TEM images (left) and mitochondria-ER distance (Interaction, F (1, 16) = 7.265, P = 0.0159), mitochondrial perimeter (Interaction, F (1, 16) = 26.96, P < 0.0001), mitochondrial area (Interaction, F (1, 16) = 29.34, P < 0.0001) and ERMICC ratio quantification (right, Interaction, F (1, 16) = 19.24, P = 0.0005) in CA1 neurons. Scale: 200 nm; Two-way ANOVA with Tukey's multiple comparisons test. Data are expressed as mean ± SEM (n = 5 mice in each group, for each mouse, all mitochondria within neuronal regions from five non overlapping hippocampal fields were analyzed, and the average value was taken as one point). ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 C) The representative images of PLA targeting IP3R3-GRP75 or GRP75-VDAC1 interaction in hippocampal CA1 (left) and the quantification of PLA red fluorescent spots (top, Interaction, F (1, 16) = 0.1194, P = 0.7342) and the percentage of PLA spots in Vglut2 (bottom, Interaction, F (1, 16) = 5.801, P = 0.0284) were performed using image J (right). Scale: 20 μm; Vglut2 (Neuron, green), nucleus (DAPI, blue). Two-way ANOVA with Tukey's multiple comparisons test. The data were expressed as mean ± SEM (n = 5 mouse brain slices). ∗∗∗ p < 0.001. D) TEM quantification of autophagosomes in CA1 (red). Two-way ANOVA with Tukey's multiple comparisons test (Interaction, F (1, 16) = 9.800, P = 0.0065). The data were expressed as mean ± SEM (n = 5 mice in each group, for each mouse, all mitochondria within neuronal regions from five non overlapping hippocampal fields were analyzed, and the average value was taken as one point). ∗ p < 0.05. E) Representative images of LAMP1 (red), rAVV (green) and DAPI (blue) fluorescence in CA1 region (left). The percentage of co-localized fluorescence was quantified (right, n = 8 mice brain slices). Scale bar: 50 or 2 μm. Two-way ANOVA with Tukey's multiple comparisons test (Interaction, F (1, 28) = 25.14, P < 0.0001). The data were expressed as mean ± SEM. ns, p > 0.05, ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: Multiplex staining was performed using TSA-based immunofluorescence with primary antibodies: Drp1 (CST 12741S, 1:100), MAP2 (ab32454, 1:1000), GFAP (CST 12389, 1:400), IBA1 (ab178846, 1:1000), Vglut1 (CST #27181, 1:800), Vglut2 (ab216463, 1:250), OLIG2 (CST #65915, 1:400), and LAMP1 (CST 99437S, 1:100).

Techniques: Knockdown, Fluorescence

Journal: Redox Biology

Article Title: Excessive ER-mitochondria coupling: A DRP1-driven mechanism underlying mitochondrial dysfunction and impaired autophagy in stress-induced depression-like behavior in mice

doi: 10.1016/j.redox.2026.104121

Figure Lengend Snippet: Drp1 regulates ER-mitochondria contacts and mitochondrial function in primary neurons. A) Schematic of primary neuronal culture preparation. B) Western blot analysis of Drp1 protein expression in primary neuron cells transfected with Drp1 siRNA or Negative control (NC) siRNA. Student's t -test (P < 0.0001). Data are expressed as mean ± SEM (n = 6 batches of transfected cell samples). ∗∗∗ p < 0.001. C) After transfection of drp1 siRNA from primary neuronal cells, the nuclei (Hoechst 33342, blue), ER (ER-tracker, green) and mitochondria (Mito-tracker, red) were stained with representative confocal images untreated (control group) or treated with CORT (400 μmol/L, left), and the co-localization between ER and mitochondria in the cell body (Interaction, F (1, 12) = 26.87, P = 0.0002) or axon (Interaction, F (1, 12) = 32.49, P < 0.0001) was quantified (right). Scale: 20 μm; Two-way ANOVA with Tukey's multiple comparisons test. The data were expressed as mean ± SEM (n = 4 batches of mouse extracted cell samples). ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. D) Representative fluorescence images, 3D thermograms (left) and quantitative analysis (right, Interaction, F (1, 12) = 10.14, P = 0.0079) of Rhod-2. Scale: 20 μm; Two-way ANOVA with Tukey's multiple comparisons test. The data were expressed as mean ± SEM (n = 4 batches of mouse extracted cell samples). ns, p > 0.05, ∗ p < 0.05, ∗∗∗ p < 0.001. E) Representative confocal images (left) of the intracellular localization of lysosome (Lyso-Tracker, red), mitochondria (Mito-Tracker, green) and nucleus (Hoechst, blue) in cells and the statistical analysis (right, n = 4 batches of mouse extracted cell samples; Interaction, F (1,12) = 35.67, P < 0.0001) of the fluorescence intensity. Scale bar: 20 μm ns, p > 0.05, ∗∗∗ p < 0.001.

Article Snippet: Multiplex staining was performed using TSA-based immunofluorescence with primary antibodies: Drp1 (CST 12741S, 1:100), MAP2 (ab32454, 1:1000), GFAP (CST 12389, 1:400), IBA1 (ab178846, 1:1000), Vglut1 (CST #27181, 1:800), Vglut2 (ab216463, 1:250), OLIG2 (CST #65915, 1:400), and LAMP1 (CST 99437S, 1:100).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Staining, Control, Fluorescence

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: ANXA3 did not regulate myocardial free fatty acid uptake or lipolysis but positively modulated macroautophagy in T2DM mice. (A-C) Relative p62 protein expression level and LC3 II/I ratio in heart tissues of db/m and db/db mice analyzed by Western blot, n = 4. (D-F) Relative p62 protein expression level and LC3 II/I ratio in heart tissues of mice analyzed by Western blot, n = 4. (G-I) Relative p62 protein expression level and LC3 II/I ratio in AC16 cardiomyocytes analyzed by Western blot, n = 6. (J-L) Relative p62 protein expression level and LC3 II/I ratio in AC16 cardiomyocytes analyzed by Western blot, n = 6-8. The data were presented as the mean ± SD. ∗ P < 0.05 versus the db/m group or the db/db + OE-GFP group; ∗∗ P < 0.01 versus the db/m group or the vehicle group or the OE-NC + vehicle group; ∗∗∗ P < 0.001 versus the vehicle group or the OE-NC + vehicle group; # P < 0.05 versus the OE-NC + HGPA group; ### P < 0.001 versus the OE-NC + HGPA group.

Article Snippet: Blocked with 5% (w/v) non-fat-dried milkat room temperature for 1 h. Then the membranes were incubated with the anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:1000 [v/v]), the anti-YY1 antibody (Proteintech, 22156-1-AP; 1:1000 [v/v]) the anti-PLIN2 antibody (Proteintech, 15294-1-AP; 1:1000 [v/v]), the anti-SQSTM1/p62 antibody (Cell Signaling Technology, 23214; 1:1000 [v/v]), the anti-LC3 antibody (Cell Signaling Technology, 12741; 1:1000 [v/v]), the anti-Rab7 antibody (Proteintech, 55469-1-AP; 1:1000 [v/v])or the rabbit anti-α-Tubulin antibody (ABclonal, AC031; 1:1000 [v/v])overnight at 4 °C.

Techniques: Expressing, Western Blot