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rabbit anti-atp5o polyclonal antibody  Rabbit Anti Atp5o Polyclonal Antibody, supplied by Brickell Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more https://www.bioz.com/result/rabbit anti-atp5o polyclonal antibody/product/Brickell Biotech Average 90 stars, based on 1 article reviews
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2026-02
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Journal: Research
Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress
doi: 10.34133/2022/9834963
Figure Lengend Snippet: Upregulated HDAC2 phosphorylation is connected with downregulated ATP5O crotonylation in CS mice. (a, b) TMT-labeled phosphoproteomics showed that at the threshold of ≥1.2 or ≤ 0.833 , there are 117 downregulated and 60 upregulated phosphorylated proteins. (c) KEGG pathway showed that multiple pathways are lipid metabolism related. (d) We selected three deacetylases—HDAC2, KAT7, and CREBBP—to verify their relationship with ATP5O. Western blot showed that inhibition of HDAC2 phosphorylation at S424 with THP (CYGRKKRRQRRRSDSEDEGEGGRR) significantly decreased ATP5O, whereas inhibition of KAT7 phosphorylation at S102 with TKP (CYGRKKRRQRRRSSGSETEQVVDF) or CREBBP phosphorylation at S2361 and S2363 with TCP (CYGRKKRRQRRRQSQPPHSSPSPR) did not change ATP5O protein level at all. (e, f) We generated an ATP5O-S424 phosphorylation-specific antibody with a phosphorylated peptide SDS (phospho) EDEGEGGRR and verified the specificity through two ways. (e) Different amounts of non- and S424-phosphorylated HDAC2 peptide were loaded onto a dot blot PVDF membrane and detected by the HDAC2-S424 P antibody. Only phosphorylated HDAC2 peptide was detected in a dose-dependent manner. (f) When HDAC2-S424 P antibody was preblocked by the phosphorylated HDAC2 peptide, significantly less amount of endogenous ATP5O-K51 cr was detected. (g) Western blot showed that THP treatment significantly decreased HDAC2-S424 P (p-HDAC2) level and upregulated ATP5O-K51 cr (ATP5O cr ) level, whereas did not change KAT7-S102 P (p-KAT) level. Besides, THP treatment did not alter the level of HDAC1-S421 p at all, which shared the highest similarity with THP (Supp. Figure ). (h, i) By live imaging in Sf9 cells, when only ATP5O-WT-EGFP titer was added into sf9 culture media, as time progressed, green fluorescence rapidly increased; in contrast, when both ATP5O-EGFP and HDAC2-WT-TagRFP titers were added, as time went by, the red fluorescence (HDAC2) quickly increased, whereas the increment extent of green fluorescence (ATP5O) was significantly slowed down. (j) Coimmunoprecipitation and western blot showed that HDAC2 significantly interacted with ATP5O but rarely interacted with ATP5A1 or ATP5B. (k) GST-pulldown was carried out to verify the direct binding between ATP5O and HDAC2. GST-ATP5O and his-tagged HDAC2-TagRFP were cloned, expressed, and purified in E.coli (From left, the fourth and fifth lane, red asterisk-labeled) and sf9 cells (From left, the third lane, red asterisk-labeled), respectively. SDS-PAGE showed that GST-ATP5O could significantly pull down HDAC2-TagRFP (From left, the sixth lane, red asterisk-labeled). L-N. Western blot showed that when a fixed amount of ATP5O-WT and increasing amounts of HDAC2-WT were cotransfected into 293 T cells, ATP5O crotonylation level rapidly decreased (l, n); in contrast, when a fixed amount of ATP5O-WT and increasing amounts of HDAC2-S424A (a nonphosphorylable mutant) were cotransfected into 293 T cells, the decrement extent of ATP5O crotonylation level became much smaller (m, n). (o–s) When HDAC2-Flag and ATP5O-StrepII were cotransfected into 293 T cells at an increasing dosage, ATP5O crotonylation (o, p) and ATP levels both increased (q); accordingly, FAM126A and STAT5A levels also increased in a dose-dependent manner (r, s). (t–u) Western blot showed that compared with ATP5O-transfected 293 T cells, HDAC2 and ATP5O cotransfected cells had decreased ATP5O crotonylation, whereas inhibition of proteasome activity with MG132 significantly recovered ATP5O crotonylation level. (v, w) Western blot showed that in an in vitro ubiquitination assay with the ATP5O-E3 ubiquitin ligase complex immunoprecipitated from CS ovaries, the ATP5O ubiquitination level was significantly higher than the level in control ovaries. X. Model: ATP5O crotonylation is resistant to ubiquitination, whereas ATP5O-K51 cr decrotonylated by HDAC2 is susceptible to ubiquitination and protease degradation. Tubulin or actin was used as a loading control. * indicates p < 0.05 , *** indicates p < 0.001 , **** indicates p < 0.0001 .
Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA);
Techniques: Labeling, Western Blot, Inhibition, Generated, Dot Blot, Membrane, Imaging, Fluorescence, Binding Assay, Clone Assay, Purification, SDS Page, Mutagenesis, Transfection, Activity Assay, In Vitro, Ubiquitin Assay, Immunoprecipitation
Journal: Research
Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress
doi: 10.34133/2022/9834963
Figure Lengend Snippet: ATP5O crotonylation level was the most downregulated in the ovaries and plasma of CS mice. (a) We created a chronic stress (CS) female mouse model using a restraining cylinder. The treatment lasted for 28 days, 6 hours per day. No food and water were provided during the daily treatment. (b–d) Multiple assays indicated that the CS mouse modeling was successful. (b) The weight of the CS female mice was significantly lower than control group; (c) the serum corticosterone level of the CS female mice was significantly higher than control group; and (d) the mRNA levels of multiple HPA (hypothalamic–pituitary–adrenal) axis-related genes including RFRP (RF amide-related peptide), GnRH (gonadotropin-releasing hormone 1), and GPR147 (neuropeptide FF receptor 1), had significant differences between CS and control groups. (e–h) Crotonylation was significantly downregulated in multiple tissues of CS female mice, such as hypothalamus (Hth), livers, and ovaries. (e, h) Pan-crotonylation in hypothalamus; (f, h) pan-crotonylation in liver; (g, h) pan-crotonylation in ovary. (i, k) Pan-Kcr immunofluorescence in multiple tissues, including hypothalamus (i), livers (j), and ovaries (k), showed that pan-Kcr signal in CS group was significantly lower than in control group, whereas the morphology and condition of these tissues appeared normal in either group. Pan-Kcr in green, DNA in blue. (l, m) Quantitative crotonylomics showed that at a threshold of ≥ 1.3 or ≤ 0.77 , there were 44 upregulated and 90 downregulated crotonylation sites, with the latter covering 67.2% of all differentially crotonylation sites (DCSs). (n) Among the proteins with DCSs, metabolism-related proteins represented the largest percentage (15.97%), and there were 15 metabolism-related pathways in the top 30 KEGG pathway (50%). (o, p). Among the top 10 downregulated crotonylation sites, the crotonylation level of ATP5O at K51 decreased by about 10 folds in CS ovaries than in control ovaries. (q–h) we generated an ATP5O-K51 crotonylation-specific antibody with a crotonylated peptide ASK (crotonyl)EKKLDQVEKELL and verified the specificity through two ways. (q) Different amounts of non- and K51-crotonylated ATP5O peptide were loaded onto a dot blot PVDF membrane and detected by the ATP5O-K51 cr antibody. Only crotonylated ATP5O peptide was detected in a dose-dependent manner (Figure (q)). (r, s) When ATP5O-K51 cr antibody was preblocked by the crotonylated ATP5O peptide, significantly less amount of endogenous ATP5O-K51 cr was detected (Figures (r) and (s)). (t, u) Western blot by ATP5O-K51 cr antibody verified the decrement of ATP5O crotonylation in CS ovaries. (v) Immunofluorescence showed that ATP5O-K51 cr level was significantly lower in CS ovaries than in control. ATP5O-K51 cr in green, DNA in blue. (w) ATP5O mRNA level did not change in CS ovaries. (x, y) Western blot by gross ATP5O antibody showed that ATP5O level was also significantly lower in CS ovaries than in control. (z) Immunofluorescence showed that gross ATP5O level was significantly lower in CS ovaries than in control. ATP5O in green, DNA in blue. Tubulin was used as a loading control. * Indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 .
Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA);
Techniques: Immunofluorescence, Generated, Dot Blot, Membrane, Western Blot
Journal: Research
Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress
doi: 10.34133/2022/9834963
Figure Lengend Snippet: Decreased ATP5O-K51 crotonylation conduced to reduced ATP5O stability and decreased lipid metabolism. (a–c) Transfection of ATP5O-WT or K51A (inactive mutant) plasmid (in pcDNA3.1) into 293 T cells and western blot by ATP5O-K51 cr antibody showed that ATP5O-K51A had significantly decreased crotonylation level (a, b), accordingly, ATP5O K51A-transfected 293 T cells had significantly lower cytoplasmic ATP level than ATP5O-WT-transfected 293 T cells. (d–g) Western blot showed that inhibition of ATP5O crotonylation with a competitive peptide TAC (CYGRKKRRQRRRATALYSAASKEKKL) significantly reduced ATP5O-K51 crotonylation (d, e) and gross ATP5O (f, g) levels, similar to CS treatment. (h, i). A region of Ubinuclein (UBN) shared the highest similarity with TAC peptide (Supp. Figure ); however, TAC treatment did not change UBN level at all. (j, k) Western blot showed that inhibition of ATP5O crotonylation with TAC significantly decreased STAT5A protein level. (l) TAC treatment significantly reduced liver ATP level. (m–o) Quantitative lipidome showed that at the threshold of ATP 5 O − TAC / Ctr ≥ 1.5 or ≤ 0.67 , there were 174 differentially upregulated or downregulated lipid metabolites (DULMs or DDLMs), among this, 22.41% (39/174) was phospholipid (PE & PC & PI), and 82.05% (32/39) of phospholipid was downregulated. (p) Representative downregulated phospholipid in TAC-treated group. (q–s) Blood biochemical index assay showed that multiple indexes in TAC group, including CREA, SOD, and HDL-C, were similar to CS group and significantly different from control group. Tubulin or actin was used as a loading control. * indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 , **** indicates p < 0.0001 .
Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA);
Techniques: Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Inhibition
Journal: Research
Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress
doi: 10.34133/2022/9834963
Figure Lengend Snippet: Correcting HDAC2 phosphorylation rescued abnormal lipid metabolism. (a, b) Western blot showed that inhibition of HDAC2 activity (phosphorylation) by a peptide THP (CYGRKKRRQRRRSDSEDEGEGGRR) significantly reduced the level of p-HDAC2, whereas ATP5O crotonylation and gross ATP5O levels significantly increased. (c, d) ATP levels in CS ovaries (c) and livers (d) were significantly lower than control, whereas THP treatment significantly recovers the decreased ATP levels close to control. (e) Mating test showed that the first litter size was significantly lower than control, while THP injection recovered the decreased litter size close to control. (f–h) Lipid metabolomes were analyzed for the female mouse serum of control, CS, and CS + THP group; six repeats per group. PCA plot showed that the metabolites in the CS group are completely separate from control group, with the CS + THP group partially overlapping with the control group (f). Heat map showed that at a threshold of CS + THP vs. CS ≥ 1.5 or ≤ 0.67 , there were 91 differentially upregulated or downregulated lipid metabolites (DULMs or DDLMs) that were recovered close to the control (g). And the beneficial lipid form—PE, PA, and PC—covered the second largest percentage (30 of 91, 32.97%) (g, h). (i) Representative differentially downregulated PEs, which were beneficial for physical health, are shown. (j, k) Western blot showed that STAT5A level significantly decreased in CS group, whereas THP treatment significantly recovered the STAT5A level of CS group close to control group. Tubulin was used as a loading control. * indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 , **** indicates p < 0.0001 .
Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA);
Techniques: Western Blot, Inhibition, Activity Assay, Injection
Journal: Research
Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress
doi: 10.34133/2022/9834963
Figure Lengend Snippet: Clinical female patients with high stress scores have decreased ATP5O as well as abnormal phospholipid metabolism overlapping with CS mice. (a–d) Western blots showed that the ATP5O level in the stress-prone ( 7 ≤ HAMA score ≤ 14 ) group was not significantly different from control group ( HAMA score ≤ 7 ), but ATP5O levels in the stress group ( HAMA score ≥ 14 ) were significantly lower than control group. (e) Quantification of ATP5O level showed that ATP5O level was negatively correlated with HAMA score. (f–h) Lipid metabolomes were determined for the human female serum of control, stress-prone, and stress groups, with ten repeats per group. Heat map of differentially upregulated or downregulated lipid metabolites (DULMs or DDLMs) showed that at a threshold of stress/control ≥1.5 or ≤ 0.67 , there were 62 upregulated and 155 downregulated lipid metabolites. Among these, 15.207% (33/217) was phospholipids (PE & PC & PI) and 15.207% (33/217) was lysophospholipids (LPE & LPC & LPA). Furthermore, 36.37% (12/33) of phospholipid, which are beneficial, was downregulated; and 100% (33/33) of lysophospholipids, which are detrimental, was upregulated. (i) Representative beneficial PE & PC & PA and detrimental LPE & LPC are shown. Transferrin was used as a loading control. * indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 .
Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA);
Techniques: Western Blot
Journal: Research
Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress
doi: 10.34133/2022/9834963
Figure Lengend Snippet: Model: chronic stress caused aberrant level of two types of PTMs, which reduce cytoplasmic ATP level and consequently reduced beneficial phospholipids. Left: Under chronic stress (CS), HDAC2-S424 p was upregulated, which resulted in downregulated ATP5O-K51 cr . Downregulated ATP5O-K51 cr consequently conduced to reduced gross ATP5O level, which finally caused decreased cytoplasmic ATP level and significantly decreased oocyte quality, fertility, and beneficial phospholipids. Middle: TAC is a competitive peptide that was able to specifically downregulate ATP5O-K51 cr . Downregulated ATP5O-K51 cr consequently conduced to reduced gross ATP5O level and cytoplasmic ATP level, which finally significantly decreased oocyte quality, fertility, and beneficial phospholipids. Therefore, TAC treatment could partially recapitulate the decreased fertility and phospholipid in CS mice. Right: THP is a competitive peptide that was able to specifically downregulate the abnormally high level of HDAC2-S424 p in CS mice, which in turn recovered downregulated ATP5O-K51 cr , gross ATP5O level, and cytoplasmic ATP level close to control. Recovered ATP level finally recovered oocyte quality, fertility, and phospholipid metabolism in CS mice, Therefore, THP treatment could partially rescue the decreased fertility and phospholipid in CS mice.
Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA);
Techniques: