Journal: Cells

Article Title: Lipid Metabolic Changes and Mitochondrial Stress in Ethanol-Treated Alveolar Type II Epithelial Cells: Initial Events Leading to Alcoholic Chronic Lung Disease

doi: 10.3390/cells14221817

Figure Lengend Snippet: AMPKα signaling in AT2 cells treated with 22, 65, and 130 mM EtOH for 6 h. Representative immunoblots and corresponding bar graphs show protein expression for p-AMPKα/AMPKα ( A ), p-ACC1/ACC1 ( B ), FAS ( C ), CPT1A ( D ), p-LKB1/LKB1 ( E ), and p-CaMKKβ/CaMKKβ ( F ). Intensities normalized to β-actin (loading control). Values are expressed as Mean ± SEM ( n = 4 replicates). * p -value ≤ 0.05 with respect to the controls. ns—non-significant.

Article Snippet: Primary antibodies against acetyl CoA carboxylase 1 (ACC1; 265 kDa; Cat # 4190) and phospho (p)-ACC1 (Ser 79; 280 kDa; Cat #3661); AMPKα (62 kDa; Cat # 5831) and p-AMPKα (Thr 172) (62 kDa; Cat # 2535); Ca 2+/ calmodulin-dependent protein kinase, kinase β (CaMKKβ; 60, 50 kDa; Cat# 4436); and p-CaMKKβ (Thr286) (60, 50 kDa; Cat# 12716); liver kinase B 1 (LKB1; 54 kDa; Cat# 3050) and p-LKB1 (Ser 428) (54 kDa; Cat # 53482); fatty acid synthase (FAS; 273 kDa; Cat # 3189); and β-actin (45 kDa; Cat # 4970) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Control

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A ) Schematic diagram of the integrative approach used to identify regulators of M2-like macrophage polarization. ( B ) Ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) identified key upstream regulators of M2-like macrophage polarization. ( C ) Heatmap showing expression levels of downstream genes of STK11 in M2-like macrophages. ( D ) Circos plot showing enriched pathways from both transcriptomic and metabolomic data. Red dots represent genes, while blue dots indicate metabolites. The y axis depicts the log 2 (fold change) in IL-4–stimulated bone marrow–derived macrophages (BMDMs) compared to vehicle-treated controls. ( E ) Alteration in expression levels of genes and metabolites in the tricarboxylic acid (TCA) cycle and glutaminolysis. Bar plots depict fold changes in metabolite levels. Up-regulated genes are represented with varying intensities of red. CoA, coenzyme A; succinate-CoA ligase GDP/ADP-forming subunit alpha (SUCLG1), succinate dehydrogenase complex subunits A (SDHA), SDHB, SDHD, citrate synthase (CS), aconitase 2 (ACO2), fumarate hydrase (FH), oxoglutarate dehydrogenase (OGDH), malate dehydrogenase 1 (MDH1), isocitrate dehydrogenase1 (IDH1), glutamate dehydrogenase 1 (GLUD1), GLUD2, glutaminase (GLS), and solute carrier family 1 member 5 (SLC1A5). ( F ) Western blot analysis of phosphorylation of STK11 at serine-428 (p-STK11 S428 ), phosphorylation of signal transducer and activator of transcription 6 (STAT6) at tyrosine-641 (p-STAT6 T641 ), and β-actin expression in BMDMs stimulated with vehicle or IL-4 for the indicated time points. Data are representative of one experiment [(B) to (E)] or at least three independent experiments (F) and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test (E). *** P < 0.001 and **** P < 0.0001. h, hours.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Expressing, Derivative Assay, Western Blot, Phospho-proteomics, Two Tailed Test

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A and B ) Relative expression of Mrc1 and Arg1 mRNA (A), and Il10 and Tgfb mRNA (B) in WT and LysM Cre Stk11 fl/fl BMDMs stimulated with IL-4 (20 ng/ml) for 24 hours. ( C to E ) Comparisons of CD206 (C), CD163 (D), and CD301 (E) expression on IL-4–stimulated WT and LysM Cre Stk11 fl/fl BMDMs. Numbers in graphs indicate the mean fluorescence intensity (MFI). ( F and G ) Relative expression of Mrc1 and Arg1 mRNA (G), and Il10 and Tgfb mRNA (H) in WT and LysM Cre Stk11 fl/fl PMs stimulated with IL-4 (20 ng/ml) for 24 hours. ( H to J ) Percentages of CD4 + T cells producing IFN-γ (H), or IL-17 (I), or IL-4 (J) in the spleen from WT and LysM Cre Stk11 fl/fl mice ( n = 5 per group). ( K ) Percentages of IFN-γ–producing CD8 T cells in the spleen from WT and LysM Cre Stk11 fl/fl mice ( n = 5 per group). ( L ) Representative images of hematoxylin and eosin staining of the lung, small intestine, and colon from WT and LysM Cre Stk11 fl/fl mice (scale bar, 100 μm). Data are representative of at least three independent experiments [(A) to (L)] and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(A) to (K)]. * P < 0.05 and ** P < 0.01; n.s., not significant.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Expressing, Fluorescence, Staining, Two Tailed Test

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A to C ) Percentages and numbers of T reg cells (Foxp3 + CD4 + ) in lymphoid tissues (A), lung (B) and liver (C) from WT and LysM Cre Stk11 fl/fl mice ( n = 5 per group). ( D ) Comparison of Ki67 expression in splenic T reg cells from WT and LysM Cre Stk11 fl/fl mice ( n = 5 per group). Numbers in graphs indicate the MFI of Ki67. ( E ) Percentages of CD103 + T reg cells in the spleen from WT and LysM Cre Stk11 fl/fl mice ( n = 5 per group). ( F and G ) Flow cytometry analysis of Foxp3 + (F) and CD103 + Foxp3 + (G) populations in OT-II CD4 + T cells cocultured with WT or LysM Cre Stk11 fl/fl BMDMs in the presence of OVA and TGF-β for 4 days. ( H and I ) Flow cytometry analysis of Foxp3 + (H) and CD103 + Foxp3 + (I) populations in OT-II CD4 + T cells cocultured with WT or STK11-deficient BMDMs in the presence of OVA and TGF-β, along with IgG or IL-10 neutralizing antibodies for 4 days. Data are representative of at least three independent experiments [(A) to (I)] and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(A) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; n.s., not significant. T regs , T reg cells.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Comparison, Expressing, Flow Cytometry, Two Tailed Test

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A ) Heatmap with unsupervised hierarchical clustering of the DEGs in WT and LysM Cre Stk11 fl/fl BMDMs stimulated with vehicle or IL-4 ( n = 3 per group). ( B ) Heatmap showing the DEGs of the conditioned WT and STK11-deficient BMDMs. ( C ) Heatmap showing the expression of M2-associated markers in BMDMs. ( D ) IPA results indicating the top canonical pathways enriched in the DEGs of IL-4–stimulated STK11-deficient BMDMs. GM-CSF, granulocyte-macrophage colony-stimulating factor; GTPases, guanosine triphosphatases; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells. ( E and F ) Comparisons of PD-L1 (E) and PD-L2 (F) expression on IL-4–stimulated WT and STK11-deficient BMDMs. ( G ) Gene set enrichment analysis results showing up-regulation of OXPHOS in STK11-deficient BMDMs. NES, normalized enrichment score. FDR, false discovery rate. ( H ) Measurement of OCR in IL-4–stimulated WT and LysM Cre Stk11 fl/fl BMDMs. R/A, rotenone plus antimycin. ( I ) Spare respiratory capacity (SRC) in WT and LysM Cre Stk11 fl/fl BMDMs. ( J ) Measurement of OCR in IL-4–stimulated BMDMs in response to the indicated mitochondrial inhibitors. ( K ) Eto-sensitive OCR in IL-4–stimulated BMDMs. Data are representative of one [(A) to (D) and (G)] or at least three [(E), (F), and (H) to (K)] independent experiments and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(E), (F), (I), and (K)] or two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test [(H) and (J)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Expressing, Two Tailed Test, Comparison

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A ) IPA results showing enhanced activation of FOXO1 and FOXO3 in IL-4–stimulated STK11-deficient BMDMs. ( B ) Immunoblotting of p-FOXO1 T24 , p-FOXO3 T32 , and β-actin in BMDMs stimulated with vehicle or IL-4. ( C ) Immunoblotting of FOXO1 and β-actin expression in BMDMs stimulated with vehicle or IL-4. h, hours. ( D ) Venn diagram showing the DEGs uniquely regulated or coregulated by FOXO1 and/or FOXO3 in IL-4–stimulated BMDMs ( n = 3). ( E ) Heatmap showing the expression of the DEGs uniquely regulated by FOXO1 in IL-4–stimulated BMDMs. ( F and G ) Comparison of PD-L1 and PD-L2 expression (F) or CD206 and CD163 expression (G) on IL-4–stimulated BMDMs in the presence of vehicle or FOXO1i. ( H ) Bar graph showing Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome Pathway Database (REACTOME) pathways significantly enriched in the DEGs uniquely regulated by FOXO1 in IL-4–stimulated STK11-deficient BMDMs. SRP, signal recognition particle. ( I ) Heatmap showing the expression of FOXO1-regulated metabolic genes in IL-4–stimulated WT and STK11-deficient BMDMs ( n = 3). Data are representative of one [(A), (D), (E), (H), and (I)] or three [(B), (C), (F), and (G)] independent experiments and are presented as the means ± SEM. P values were calculated using one-way ANOVA with Tukey’s multiple comparison test [(F) and (G)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Activation Assay, Western Blot, Expressing, Comparison

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A and B ) Measurement of cellular glutamine (A) and glutamate (B) in WT and LysM Cre Stk11 fl/fl BMDMs stimulated with vehicle or IL-4. ( C ) Schematic diagram of specific inhibitors targeting the glutamine transportation and catabolism pathway. ( D to F ) Comparison of CD206 and PD-L2 expression on IL-4–stimulated WT and STK11-deficient BMDMs treated with vehicle or V-9302 (D), or BPTES (E), or R162 (F) (WT with vehicle treatment (mock) is set to 1, n = 3). ( G ) Relative fold change of α-KG/Suc ratio in IL-4–stimulated WT and STK11-deficient BMDMs. ( H ) Measurement of OCR in WT and LysM Cre Stk11 fl/fl BMDMs treated with mock or without DE-Suc in response to Oligo, FCCP, and R/A stimulation. ( I and J ) Comparison of CD206 and PD-L2 (I) and CD163 (J) expression on IL-4–stimulated WT and STK11-deficient BMDMs supplemented with mock or DE-Suc (WT with mock treatment is set to 1, n = 3). Data are representative of three independent experiments [(A), (B), and (D) to (J)] and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(A), (B), and (G)] or one-way ANOVA with Tukey’s multiple comparison test [(D) to (F), (I), and (J)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Comparison, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A to C ) Kaplan-Meier survival curves of patients with high (red) or low (blue) STK11 expression in pancreatic adenocarcinoma (PAAD) (A), uterine corpus endometrial carcinoma (UCEC) (B), and head and neck squamous cell carcinoma (HNSC) (C). ( D ) Experimental design for in vivo imaging system (IVIS) imaging of mice orthotopically inoculated Luc + KPC tumor cells. ( E ) Bioluminescent images of WT and LysM Cre Stk11 fl/fl mice bearing orthotopically inoculated Luc + KPC tumors ( n = 5 per group). ( F ) Bioluminescent analysis of KPC tumor growth in WT and LysM Cre Stk11 fl/fl mice. ( G ) Growth of KPC tumors in WT and LysM Cre Stk11 fl/fl individuals based on bioluminescent analysis ( n = 5 per group). ( H ) Survival curves of the KPC tumor-bearing WT and LysM Cre Stk11 fl/fl mice ( n = 5 per group). ( I ) Representative images of KPC tumors from WT and LysM Cre Stk11 fl/fl mice ( n = 4 per group). ( J and K ) KPC tumor volume (J) and weight (K) from WT and LysM Cre Stk11 fl/fl mice in (I). Data are from one experiment [(A) to (C) and (E) to (H)] or representative of two-independent experiments [(I) and (K)] and are presented as means ± SEM. P values were calculated using Log-rank test [(A) to (C) and (H)], two-way ANOVA with Bonferroni’s multiple comparison test (F), or two-tailed Student’s t test [(J) and (K)]. * P < 0.05, ** P < 0.01, and ****P < 0.0001.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Expressing, In Vivo Imaging, Imaging, Comparison, Two Tailed Test

Journal: Science Advances

Article Title: STK11 coordinates IL-4 signaling with metabolic reprogramming to control M2 macrophage polarization and antitumor immunity

doi: 10.1126/sciadv.adx5495

Figure Lengend Snippet: ( A ) t -SNE analysis (left) and stacked bar graph (right) of CD11B + cell populations in KPC tumors from WT and LysM Cre Stk11 fl/fl mice ( n = 4 per group). ( B ) Percentages of eosinophils (Siglec-F + CD11b + ) in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. ( C and D ) Percentages of F4/80 + CD206 + (C) and F4/80 + PD-L2 + (D) M2-like TAMs in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. ( E ) t -SNE analysis of IL-10–producing CD11b + myeloid cells (left) and percentages of IL-10–producing macrophages (right) in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. ( F ) t -SNE analysis (left) and stacked bar graph (right) of CD11b − cell populations in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. ( G and H ) Percentages of IOCS + T reg cells (G) and KLRG1 + T reg cells (H) in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. ( I ) Flow cytometry analysis (left) and percentages (right) of PD-1–expressing CD8 + T cells in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. ( J ) Percentages of IFN-γ–expressing CD8 T cells in KPC tumors from WT and LysM Cre Stk11 fl/fl mice. Data are representative of two independent experiments [(A) to (J)] and are presented as means ± SEM. P values were calculated using two-tailed Student’s t test [(B) to (D) and (G) to (J)]. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Immunoblots were performed as described previously, using the following antibodies: p-LKB1 S428 , p-AKT S473 , p-4E-BP1 T37/46 , p-STAT6 Tyr641 , p-FOXO1 Thr24 /FOXO3 Thr32 , FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology).

Techniques: Flow Cytometry, Expressing, Two Tailed Test

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 8-Prenylnaringenin suppresses obesity in high-fat diet-fed C57BL/6J mice via adiponectin secretion

doi: 10.3164/jcbn.24-214

Figure Lengend Snippet: Effects of 8-PN on protein levels and phosphorylation of LKB1 and CaMKKβ. (A, B) Protein levels and phosphorylation levels of (A) LKB1 and (B) CaMKKβ are shown. Tissue lysate was extracted from the epididymal white adipose tissue of mice fed a control or high-fat diet with or without 8-PN. Data are presented as mean ± SE ( n = 5–7). Means were compared among the six groups using the Tukey–Kramer multiple comparison test; means with the same letter(s) are not significantly different from each other. The significance level was set as p <0.05. 8-PN, 8-prenylnaringenin; LKB1, liver kinase B1; CaMKKβ, calcium/calmodulin-activated kinase kinase β.

Article Snippet: Primary antibodies for western blotting [PGC-1α (#2178S), AMPK (#2532S), p-liver kinase B1 (LKB1) (#3055S), LKB1 (#3047S), p-calcium/calmodulin-activated kinase kinase β (CaMKKβ) (#12818S), PPARγ (#2443S), and β-actin (#4967S)], and secondary antibody horseradish peroxidase-conjugated anti-rabbit immunoglobin G (#7074), were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Control, Comparison

Journal: Advanced Science

Article Title: The GRK2/AP‐1 Signaling Axis Mediates Vascular Endothelial Dysfunction and Atherosclerosis Induced by Oscillatory Low Shear Stress

doi: 10.1002/advs.202501981

Figure Lengend Snippet: NR4A1 functions in vascular endothelial cell energy metabolism disorders induced by OSS by binding to LKB1 in the nucleus. a,b) Co‐IP experiment revealing the direct binding of NR4A1 to LKB1 in HEK293 cells (n = 3). c) Co‐IP experiment revealing the direct binding of NR4A1 to LKB1 in HUVECs (n = 3). d) Immunofluorescence staining for LKB1 (green) and DAPI (blue) in HUVECs exposed to different shear stresses. e) NR4A1 siRNA treated‐HUVECs and scramble siRNA treated‐HUVECs were exposed to OSS and LSS, and the LKB1 protein expression levels in the nucleus and cytoplasm of the HUVECs were detected by Western blotting. f) Quantification of LKB1 protein expression levels in the nucleus and cytoplasm (n = 5, one‐way ANOVA). g) Immunofluorescence staining for LKB1 S428p (green) and DAPI (blue) in NR4A1 siRNA treated‐HUVECs and scramble siRNA treated‐HUVECs exposed to different shear stresses. h) Quantification of LKB1 immunofluorescent intensity (n = 5, one‐way ANOVA). i) NR4A1 siRNA treated‐HUVECs and scramble siRNA treated‐HUVECs were subjected to different shear stresses, and the protein expression levels of LKB1 S428p , LKB1, AMPK T172p , and AMPK were detected by Western blotting. j) Quantification of LKB1 S428p and AMPK T172p protein expression levels (n = 5, one‐way ANOVA). k) eNOS S1177p expression in constitutively activated LKB1 S428D ‐overexpressing HUVECs and vector control‐transfected HUVECs exposed to different shear stresses was detected via Western blotting. l) Quantification of eNOS S1177p protein expression levels (n = 5, one‐way ANOVA). m) ROS levels were detected by DCFH‐DA (green) in constitutively activated LKB1 S428D ‐overexpressing HUVECs and vector control HUVECs exposed to different shear stresses. n) Quantification of ROS levels (n = 5, one‐way ANOVA). o) Extracellular acidification rate (ECAR) profiles showing glycolytic function in vector‐, GRK2 S29D ‐, AP‐1 S63D ‐, and NR4A1‐overexpressing HUVECs. The vertical lines indicate the time of addition of glucose (10 mmol L −1 ), oligomycin (3 µmol L −1 ), and 2‐deoxy‐D‐glucose (2‐DG) (100 mmol L −1 ). p) Quantification of glycolytic function parameters from (o); values are normalized to those of 10⁴ cells (n = 5, one‐way ANOVA). q) Oxygen consumption rate (OCR) profiles showing mitochondrial respiration function in vector‐, GRK2 S29D ‐, AP‐1 S63D ‐, and NR4A1‐overexpressing HUVECs. The vertical lines indicate the time of addition of oligomycin (3 µmol L −1 ), trifluoromethoxy phenylhydrazone (FCCP) (1 µmol L −1 ), antimycin A (1.5 µmol L −1 ), or rotenone (3 µmol L −1 ). r) Quantification of mitochondrial respiration function parameters from (q); values normalized to those of 10⁴ cells (n = 5, one‐way ANOVA).

Article Snippet: The antibodies used included GRK2 (Phospho‐Ser29) (EnoGene, #E11‐0486A, New York, New York, USA), GRK2 (Phospho‐Tyr13) (Thermo Fisher, #PA5‐64755, Waltham, Massachusetts, USA), GRK2 (Phospho‐Tyr86) (Thermo Fisher, #PA5‐64756, Waltham, Massachusetts, USA), GRK2 (Phospho‐Ser685) (Immunoway, #YP1225, Newark, Delaware, USA), GRK2 (Phospho‐Ser670) (Affinity Bioscience, #AF3697, Nanjing, Jiangsu, China), GRK2 (Santa Cruz, #sc‐13143, Dallas, Texas, USA), GRK3 (Abclonal, #A9163, Wuhan, Hubei, China), GRK4 (Abclonal, #A10370, Wuhan, Hubei, China), GRK5 (Abclonal, #A3899, Wuhan, Hubei, China), GRK6 (Abclonal, #A6379, Wuhan, Hubei, China), CD31 (Immunoway, # PT0350R, Newark, Delaware, USA), α‐SMA (Immunoway, # YM6566, Newark, Delaware, USA), AP‐1 (Phospho‐Ser63) (Abclonal, #AP0105, Wuhan, Hubei, China), AP‐1 (Abclonal, #A11378, Wuhan, Hubei, China), NR4A1 (Santa Cruz, #sc‐365113, Dallas, Texas, USA), LKB1 (Phospho‐Ser428) (Santa Cruz, #sc‐271924, Dallas, Texas, USA), LKB1 (Santa Cruz, #sc‐32245, Dallas, Texas, USA), AMPK (Phospho‐Tyr172) (Abclonal, #AP1441, Wuhan, Hubei, China), AMPK (Abclonal, #A12718, Wuhan, Hubei, China), VCAM‐1 (Proteintech, #66294‐1‐Ig, Wuhan, Hubei, China), ICAM‐1 (Abclonal, #A19300, Wuhan, Hubei, China), eNOS (Phospho‐Ser1177) (Santa Cruz, #sc‐81510, Dallas, Texas, USA), and GAPDH (Abclonal, #A19056, Wuhan, Hubei, China).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Shear, Expressing, Western Blot, Plasmid Preparation, Control, Transfection

Journal: Advanced Science

Article Title: The GRK2/AP‐1 Signaling Axis Mediates Vascular Endothelial Dysfunction and Atherosclerosis Induced by Oscillatory Low Shear Stress

doi: 10.1002/advs.202501981

Figure Lengend Snippet: Schematic illustration. OSS activates the GPCR in vascular endothelial cells and leads to the phosphorylation of GRK2, which in turn phosphorylates the downstream transcription factor AP‐1. AP‐1 promotes the transcription of the monocyte recruitment factors ICAM1 and VCAM1, causing inflammation in the vascular endothelium. In addition, AP‐1 also leads to increased expression levels of NR4A1, which binds with LKB1 in the nucleus, resulting in decreased LKB1 phosphorylation and activity. This ultimately leads to AMPK inactivation, oxidative stress, and reductions in eNOS activity.

Article Snippet: The antibodies used included GRK2 (Phospho‐Ser29) (EnoGene, #E11‐0486A, New York, New York, USA), GRK2 (Phospho‐Tyr13) (Thermo Fisher, #PA5‐64755, Waltham, Massachusetts, USA), GRK2 (Phospho‐Tyr86) (Thermo Fisher, #PA5‐64756, Waltham, Massachusetts, USA), GRK2 (Phospho‐Ser685) (Immunoway, #YP1225, Newark, Delaware, USA), GRK2 (Phospho‐Ser670) (Affinity Bioscience, #AF3697, Nanjing, Jiangsu, China), GRK2 (Santa Cruz, #sc‐13143, Dallas, Texas, USA), GRK3 (Abclonal, #A9163, Wuhan, Hubei, China), GRK4 (Abclonal, #A10370, Wuhan, Hubei, China), GRK5 (Abclonal, #A3899, Wuhan, Hubei, China), GRK6 (Abclonal, #A6379, Wuhan, Hubei, China), CD31 (Immunoway, # PT0350R, Newark, Delaware, USA), α‐SMA (Immunoway, # YM6566, Newark, Delaware, USA), AP‐1 (Phospho‐Ser63) (Abclonal, #AP0105, Wuhan, Hubei, China), AP‐1 (Abclonal, #A11378, Wuhan, Hubei, China), NR4A1 (Santa Cruz, #sc‐365113, Dallas, Texas, USA), LKB1 (Phospho‐Ser428) (Santa Cruz, #sc‐271924, Dallas, Texas, USA), LKB1 (Santa Cruz, #sc‐32245, Dallas, Texas, USA), AMPK (Phospho‐Tyr172) (Abclonal, #AP1441, Wuhan, Hubei, China), AMPK (Abclonal, #A12718, Wuhan, Hubei, China), VCAM‐1 (Proteintech, #66294‐1‐Ig, Wuhan, Hubei, China), ICAM‐1 (Abclonal, #A19300, Wuhan, Hubei, China), eNOS (Phospho‐Ser1177) (Santa Cruz, #sc‐81510, Dallas, Texas, USA), and GAPDH (Abclonal, #A19056, Wuhan, Hubei, China).

Techniques: Phospho-proteomics, Expressing, Activity Assay