Journal: Cancer cell

Article Title: Integrative multi-omic cancer profiling reveals DNA methylation patterns associated with therapeutic vulnerability and cell-of-origin.

doi: 10.1016/j.ccell.2023.07.013

Figure Lengend Snippet: Figure 5. STAT5A hypermethylation associated with pervasive STAT5A regulon changes (A) Unsupervised clustering of STAT5A regulon genes using Pearson correlation of scaled RNA sequencing data. Annotations denote STAT5A expression and methylation levels. Mean activity indicates the overall sum of regulon activity. The color scale is proportional to expression (red: upregulation; blue: down- regulation). (B) Unsupervised clustering of STAT5A regulon genes using Pearson correlation of scaled global proteome data. (C) Violin plot comparing regulon activity in hypermethylated STAT5A (yellow) and normally methylated STAT5A (gray) samples. Median values are shown as solid black lines, and first and third quartiles are represented by dashed lines. Statistical significance was determined using a Wilcoxon rank-sum test. (D) Pathway members and interactions in the STAT5A regulon. The mean expression differences between STAT5A hypermethylated samples and normally methylated samples are indicated by shading of the filled squares. See also Figure S5 and Table S3.

Article Snippet: Staining employed the Dako Autostainer Link 48 with EnVision FLEX visualizing kit (K800221-2; Dako, Agilent Technologies Inc.) and rabbit polyclonal antibody against human STAT5A (HPA042128, Atlas Antibodies, 1:150 dilution).

Techniques: RNA Sequencing, Expressing, Methylation, Activity Assay

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); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

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: Lipid metabolism was downregulated in the plasma of CS mice. (a). Liver ATP level was significantly lowers in CS group than in control. (b). Full-spectrum metabolome was performed on the female mouse serum of CS and control groups, six repeats per group. PCA (principal component analysis) plot showed that the metabolites in CS group were fairly distinct from control group. (c, d) Volcano Plot and Heat map showed that at a threshold of CS/control ≥ 1.5 or ≤ 0.67 , there were 435 downregulated and 189 upregulated metabolites. (e) Pie charts showed differential metabolite species, 68.91% (430/624) of the differential metabolites were lipids. Among the differential lipid metabolites, the first three categories are TG (triglycerides); PC & PE & PI (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol); and LPC & LPE (lysophosphatidylcholine, lysophosphatidylethanolamine). (f) Representative differentially downregulated PC and PE, which were beneficial for physical health, and representative differentially upregulated LPC and LPE, which were detrimental for physical health, are shown. (g–l). Western blot showed that among three lipid-metabolism-related enzymes, including STAT5A, FAM126A, and PTDSS1, STAT5A significantly decreased in the CS hypothalamus (Hth) (g, h), livers (i, j), and ovaries (k, l), whereas PTDSS1 and FAM126A significantly decreased only in CS ovaries. (m–o) Switching the 293 T cell culture media from high- to low-glucose DMEM significantly decreased cytoplasmic ATP level (m) as well as STAT5A level (n, o). (p–x) Blood biochemical index showed that multiple indexes, including TG (triglyceride), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREA (creatinine), TP (total protein), ALP (alkaline phosphatase), and UA (Uric Acid), were significantly different between CS and control groups. (y) Blood biochemical index assay showed that a month past CS treatment, LDL-C level in CS group was still significantly higher than control. 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); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Western Blot, Cell Culture

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); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

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); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Western Blot, Inhibition, Activity Assay, Injection