h3 cst 4620s  (Cell Signaling Technology Inc)

Journal: The Journal of Headache and Pain

Article Title: S100A11 regulates microglial inflammatory response in neuropathic pain via H3K27ac-TFEB-mitochondrial autophagy axis

doi: 10.1186/s10194-026-02328-9

Figure Lengend Snippet: S100A11 knockdown alleviates microglial inflammation by inhibiting mitophagy through the TFEB/H3K27ac axis. A-B Western blot analysis of TFEB, TFEC, and TFE3 protein expression levels in the cytoplasm and nucleus of primary microglia, along with the corresponding detection and quantification plots ( D-F , for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). C Immunofluorescence staining of TFEB in primary microglia. G Western blot analysis of pink1, Parkin, P62, LC3II protein levels of primary microglia; H-K Relative levels normalized to COX IV were quantified (for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). M Labeling of mitochondria (red) in primary microglia with Tom20; immunofluorescence staining for LC3B (green). Quantification of the mean fluorescence intensity of LC3 is shown in panel L . and relative fluorescence intensity was quantified in panel L ( n = 3). N Western blot analysis of H4K16ac, H4K12ac, H3K27ac, H3K18ac, H3K9ac protein levels of microglia; O-S Relative levels normalized to histone H3 were quantified (for three independent experiments, statistical significance was determined by two-way ANOVA with appropriate post hoc tests). T Western blot analysis of Pink1, Parkin, P62, LC3II protein levels of primary microglia; U-X Relative levels normalized to COX IV were quantified (for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). Z Labeling of mitochondria (red) in primary microglia with Tom20; immunofluorescence staining for LC3B (green), and relative fluorescence intensity was quantified in panel Y ( n = 3). Scale bars as indicated. Data are presented as mean ± SEM. * P < 0.05 , ** P < 0.01 , *** P < 0.001

Article Snippet: Membranes were blocked with 5% skim milk for 60 min, rinsed with TBST, and incubated with primary antibodies at 4 °C overnight (β-actin: Proteintech, 66009-1-Ig, 1:10000; S100A11: Abcam, ab169530, 1:1000; NLRP3: AdipoGen, AG-20B-0014, 1:1000; GSDMD-N: Abcam, ab215203, 1:1000; ASC: Wanleibio, WL02462, 1:500; Caspase1: CST, 89332, 1:1000; Bax: Proteintech, 50599-2-Ig, 1:2000; Bcl-2: abclone, A19693, 1:2000; COXIV: Proteintech, 11242-1-AP, 1:1000; LC3: Proteintech, 14600-1-AP, 1:2000; P62: Proteintech, 18420-1-AP, 1:2000; Parkin: Proteintech, 14060-1-AP, 1:1000; Pink1: Proteintech, 23274-1-AP, 1:2000; H3: Proteintech, 17168-1-AP, 1:2000; TFEB: Proteintech, 13372-1-AP, 1:1000; TFEC: Proteintech, 13547-1-AP, 1:1000; TFE3: Proteintech, 14480-1-AP, 1:1000; H3K9ac: CST, 9649, 1:1000; H3K18ac: CST, 13998, 1:1000; H3K27ac: CST, 8173, 1:1000; H4K12ac; CST, 13944, 1:1000; H4K16ac: CST, 13534, 1:1000).

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Labeling, Fluorescence

Journal: The Journal of Headache and Pain

Article Title: S100A11 regulates microglial inflammatory response in neuropathic pain via H3K27ac-TFEB-mitochondrial autophagy axis

doi: 10.1186/s10194-026-02328-9

Figure Lengend Snippet: S100A11 knockdown alleviates microglial inflammation by inhibiting mitophagy through the TFEB/H3K27ac axis. A-B Western blot analysis of TFEB, TFEC, and TFE3 protein expression levels in the cytoplasm and nucleus of primary microglia, along with the corresponding detection and quantification plots ( D-F , for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). C Immunofluorescence staining of TFEB in primary microglia. G Western blot analysis of pink1, Parkin, P62, LC3II protein levels of primary microglia; H-K Relative levels normalized to COX IV were quantified (for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). M Labeling of mitochondria (red) in primary microglia with Tom20; immunofluorescence staining for LC3B (green). Quantification of the mean fluorescence intensity of LC3 is shown in panel L . and relative fluorescence intensity was quantified in panel L ( n = 3). N Western blot analysis of H4K16ac, H4K12ac, H3K27ac, H3K18ac, H3K9ac protein levels of microglia; O-S Relative levels normalized to histone H3 were quantified (for three independent experiments, statistical significance was determined by two-way ANOVA with appropriate post hoc tests). T Western blot analysis of Pink1, Parkin, P62, LC3II protein levels of primary microglia; U-X Relative levels normalized to COX IV were quantified (for three independent experiments, statistical significance was determined by one-way ANOVA with appropriate post hoc tests). Z Labeling of mitochondria (red) in primary microglia with Tom20; immunofluorescence staining for LC3B (green), and relative fluorescence intensity was quantified in panel Y ( n = 3). Scale bars as indicated. Data are presented as mean ± SEM. * P < 0.05 , ** P < 0.01 , *** P < 0.001

Article Snippet: Membranes were blocked with 5% skim milk for 60 min, rinsed with TBST, and incubated with primary antibodies at 4 °C overnight (β-actin: Proteintech, 66009-1-Ig, 1:10000; S100A11: Abcam, ab169530, 1:1000; NLRP3: AdipoGen, AG-20B-0014, 1:1000; GSDMD-N: Abcam, ab215203, 1:1000; ASC: Wanleibio, WL02462, 1:500; Caspase1: CST, 89332, 1:1000; Bax: Proteintech, 50599-2-Ig, 1:2000; Bcl-2: abclone, A19693, 1:2000; COXIV: Proteintech, 11242-1-AP, 1:1000; LC3: Proteintech, 14600-1-AP, 1:2000; P62: Proteintech, 18420-1-AP, 1:2000; Parkin: Proteintech, 14060-1-AP, 1:1000; Pink1: Proteintech, 23274-1-AP, 1:2000; H3: Proteintech, 17168-1-AP, 1:2000; TFEB: Proteintech, 13372-1-AP, 1:1000; TFEC: Proteintech, 13547-1-AP, 1:1000; TFE3: Proteintech, 14480-1-AP, 1:1000; H3K9ac: CST, 9649, 1:1000; H3K18ac: CST, 13998, 1:1000; H3K27ac: CST, 8173, 1:1000; H4K12ac; CST, 13944, 1:1000; H4K16ac: CST, 13534, 1:1000).

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Labeling, Fluorescence