EMBO/soda-nel huggingface dataset:
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 1

Figure 1 Phenotypic changes in adipocyte-specific QKI-deficient mice.

(A)The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice (n = 8) and lean controls (n = 8).

(B) WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed a NCD or HFD for 20 weeks. The arrow indicates the time when the high fat diet（HFD）starts, and body weight was monitored weekly. (n = 10, per genotype)

(C) Representative pictures of adipocyte-specific QKI-deficient mice under NCD or HFD conditions.

(D) Gross appearance of tissues from BAT, eWAT and ingWAT fat pads from WT fl/fl and QKI KO fl/fl:AP2-Cre mice after 20 weeks on the NCD or HFD.

(E) H&E staining of BAT, eWAT and ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)

(F) The workflow for administration of AAV-ShQKI or AAV-NC vectors into BAT and ingWAT to suppress QKI expression under HFD conditions.

(G) The gross appearance of BAT, eWAT and ingWAT fat pads after administration of AAV-ShQKI or AAV-NC vectors in local adipose (ingWAT and BAT).

(H) The increase in relative body weights following HFD feeding. (n = 8, per group)

(I) H&E staining of the liver, BAT and ingWAT from AAV-NC or AAV-ShQKI mice. (Scale bar represents 50 μm).

Data information: All data are presented as the mean ± SEM; a two-tailed Student's t-test (A) and two-way ANOVA (B, H) were used for statistical analyses. n.s. not significant, *p < 0.05, **p < 0.01.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 2

Figure 2 Increased insulin sensitivity in adipose-specific QKI-deficient mice.

(A) Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition.

(B, C) Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT (B) and ITT (C) were monitored. (n= 10, per genotype)

(D) Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)

(E) Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)

(F) H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)

Data information: All data are presented as the mean ± SEM, and statistical significance was determined by two-way ANOVA (B, C) and two-tailed Student's t tests (D, E, F). n.s. not significant, *p < 0.05, **p < 0.01.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 3

Figure 3 Enhanced thermogenesis in QKI-deficient mice.

(A, B) O2 consumption rates were measured via indirect calorimetry using CLAMS under chow-fed conditions (n = 8, per genotype). Heat production rates in WT fl/fl and QKI KO fl/fl:AP2-Cre mice (n = 8, per genotype). And average light and dark O2 consumption and heat production rates are shown on the right.

(C) Heatmaps of log2 (fold change QKI KO fl/fl:AP2-Cre BAT vs. WT fl/fl BAT) for genes involved in thermogenesis and oxidation. (3 samples, per group)

(D) Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice.

(E) The average diameter of lipid droplets and the number of mitochondria per field from WT fl/fl and QKI KO fl/fl:AP2-Cre BAT. (n = 3 biological replicates)

(F) The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT-PCR. (n = 4 biological replicates)

(G) Total oxygen consumption rates (OCR) per gram in BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions were measured using a Clark electrode. (n = 3 biological replicates)

(H) The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT-PCR. (n = 4 biological replicates)

(I) Total oxygen consumption rates (OCR) per gram in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions. (n = 3 biological replicates)

(J) Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP.

(K) Quantification of experimental data. (n=3 biological replicates, per group)

(L) UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)

Data information: All data are presented as the mean ± SEM. A two-tailed Student's t-test was used for statistical analysis. n.s. not significant, *p < 0.05, **p < 0.01.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 4

Figure 4 QKI is a repressor of PGC1α to modulate adipocyte metabolic status.

(A) Venn diagram demonstrating the significant enrichment of QKI-mediated change genes in the set of QKI binding genes determined by RIP-seq experiment. Only 30 genes identified by microarray analysis were also QKI binding targets.

(B) Schematic diagram of search strategy to identify genes, which contained QRE motif sites and involved in controlling adipose thermogenic metabolism. The following publicly available datasets were used for the comparisons: brown versus white adipose tissues (WAT) (NCBI GEO: GSE51080), BAT following cold exposure

(NCBI GEO: GSE40486) and WAT following cold exposure (NCBI GEO: GSE51080).

(C) The protein levels of PGC1α and QKI in siRNA-mediated QKI knockdown differentiated cell, and the quantification graph was shown on the right. (n=3 biological replicates)

(D) Immunostaining of PGC1α and UCP1 in NC, Si-QKI 1, and Si-QKI 2 adipocytes. (Scale bar represents 20 μm).

(E) The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)

(F) The oxygen consumption rates in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)

(G) Representative pictures of oil red O staining in three adipocyte types: ShScramble+ovCon cells, QKI knockdown cells (ShQKI+ovCon) and QKI knockdown with QKI-5 re-expression cells (ShQKI+ovQKI).

(H) The protein levels of PGC1α in these adipocytes, and the quantification graph was shown on the right. (n=3 biological replicates)

(I) The relative mRNA levels of genes associated with energy dissipation in ShScramble+ovCon, ShQKI+ovCon and ShQKI+ovQKI differentiated cells. The results represent 3 independent experiments.

Data information: All data are presented as the mean ± SEM. One-way ANOVA with Tukey's comparison test was used for statistical analysis. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 5

Figure 5 QKI specifically binds to the QRE motif sites in the PGC1α and UCP1 3'UTR regions.

(A) Construction of a series of luciferase reporter vectors (PGC1α full-length 3'UTR T, A, B, C fragments and PGC1α 3'UTR QRE motif sites mutant B M1, B M2, C M1, C M2 fragments). Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3'UTR affected by QKI. (n=4 biological replicates)

(B) Construction of a series of luciferase reporter vectors (UCP1 full-length 3'UTR T fragment and UCP1 3'UTR QRE motif sites mutant T M1, T M2, T M3 fragments). Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3'UTR affected by QKI. (n=4 biological replicates)

(C) Based on RIP-sequencing data, the distribution of QKI-binding peaks within UCP1 and PGC1α transcript regions.

(D) RNA immunoprecipitation followed by real-time PCR. Specific primer for PGC1α 3'UTR region 1, region 2 and UCP1 3'UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue, and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP. (n=4 biological replicates)

(E-G) 293T cells were transfected with PGC1α and UCP1 wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of PGC1α and UCP1. (n=4 biological replicates)

Data information: All data are presented as the mean ± SEM. A two-tailed Student's t-test was used for statistical analysis. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 6

Figure 6 QKI regulates thermogenic mRNA stability, nuclear export and translation efficacy.

(A-C) The mRNA stability in adipocytes was measured by incubating cells with actinomycin D (5 μg/ml). Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated times (x-axis) after transcription inhibition. Real-time PCR was used to determine remaining UCP1, PGC1α and Actin mRNA levels compared with the starting time. Increasing PGC1α mRNA half-life by depletion of QKI (ShScramble T1/2 = 4.73 h, ShQKI T1/2 = 8.04 h). (n=4 biological replicates)

(D) The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers (GAPDH and Lamin B1) to show the purity of fractions.

(E-G) Real-time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI. Values represent the cytoplasmic-to-nuclear ratio of each transcript. Actin served as a negative control. (n=4 biological replicates)

(H, I) RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation (RIP) of RNA-protein complexes using either anti-QKI antibody or control IgG. And RIP-RT-PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus. (n=3 biological replicates)

(J-L) Control (ShScramble) and QKI knockdown (ShQKI) adipocyte lysates were subjected to polysome profiling, and a representative polysome profile is shown. Total RNA was extracted from each fraction, and the distribution of UCP1 and PGC1α mRNA was measured via real-time PCR (K and L). The results are shown as the percentage of total mRNA (% mRNA) in polysome fractions and represent 3 independent experiments.

Data information: All data are presented as the mean ± SEM. A two-tailed Student's t-test was used for statistical analysis. n.s. not significant, *p < 0.05, **p < 0.01, ****p < 0.0001.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 7

Figure 7 QKI knockout mice are resistant to hypothermia upon cold stimulus.

(A) The rectal temperature of WT fl/fl and QKI KO fl/fl:AP2-Cre mice was monitored upon acute cold exposure (4 °C). (n = 10 mice, per group)

(B) H&E staining and UCP1 immunostaining of sections of ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT (22 °C) or following 72 h of cold exposure (4 °C). (Scale bar: 50 μm).

(C and D) The protein levels of UCP1 in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT or following 72 h of cold exposure (4 °C), and quantified protein levels are shown in (D). (n=3 biological replicates, per group)

(E and F) Red Nile staining and TG content in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C). (n=4 biological replicates, per group)

(G and H) The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure (4 °C) and the quantified protein levels. (n = 4 biological replicates, per group)

(I) The mRNA levels of genes related to energy consumption (PGC1α, UCP1 and Dio2). (n=4 biological replicates, per group)

Data information: All data are presented as the mean ± SEM. All data were analyzed using a two-tailed Student's t-test. n.s. not significant, *p < 0. 05, **p < 0. 01.
QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity (2019)
Figure 8

Figure 8 QKI plays a negative feedback role in cAMP signaling-induced thermogenesis.

(A, B) The protein levels of QKI in BAT and ingWAT during cold stimulation (4 °C) and the quantified protein levels. (n=3 biological replicates)

(C) The relative luminescence of a QKI promoter reporter under cAMP (Forskolin and IBMX) stimulus. (n=3 biological replicates)

(D) The effect of transcriptional factors on the QKI promoter, assessed by luciferase activity assays. (n=3 biological replicates)

(E-G) The relative mRNA levels of genes (UCP1, PGC1α, Dio2) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus, following cAMP (Forskolin and IBMX) stimulation. (n=3 biological replicates)

Data information: All data are presented as the mean ± SEM, and statistical significance was determined by a two-tailed Student's t test (E-G) and one-way ANOVA (A, B, C, D). n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced Type I IFN expression (2016)
Figure 1

Figure 1. HSV-1 harbors a mechanism to inhibit type I IFN expression, which is dependent on viral gene expression but independent of ICP0

(A) THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.

(B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(C) THP1 cells were treated with infectious or UV-inactivated HSV-1 (strain KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(D) MDMs were infected with ICP0-deficient or revertant HSV-1 (strain KOS, MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

Data are presented as means of triplicates +/- st.dev; Symbols for p-values used in the figures: *, 0.01
HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced Type I IFN expression (2016)
Figure 2

Figure 2. HSV-1 does not replicate productively in macrophages, but ICP27 expression correlates inversely with Type I IFN production

(A) MDMs were infected for 18 h with HSV-1 (KOS, MOI 1). Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.

(B) MDMs were infected with the KOS or F strains of HSV-1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β-actin were determined by Western blotting.

(C) HFFs and MDMs were infected with HSV-1 (KOS, MOI 0.1 and 1.0). Culture medium was replaced 3 hpi, and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates +/- st.dev.

(D) THP1 cells were infected with the indicated strains of HSV-1 (MOI 3) for 12 hrs. Cell lysates were isolated and levels of ICP27 and β-actin were determined by Western blotting.
HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced Type I IFN expression (2016)
Figure 3

Figure 3. ICP27 inhibits the cGAS-STING pathway in macrophages

(A) THP1 cells were infected with HSV-1 KOS or an ICP27-deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.

(B) MDMs were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(C) THP1-derived cells deficient for cGAS or STING were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(D) HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2x104 cells per well). Reporter gene activity was measured in lysates isolated 24 h post transfection.

(E) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity

Data are presented as means of triplicates +/- st.dev; Symbols for p-values used in the figures: *, 0.01
HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced Type I IFN expression (2016)
Figure 4

Figure 4. ICP27 inhibits the cGAS-STING pathway downstream of TBK1 phosphorylation but upstream of IRF3 phosphorylation

(A) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting.

(B) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 10). The cells were fixed at the indicated time points post infection, and stained with DAPI and an antibody against ICP27, and visualized by confocal microscopy. Scale bar 10 μm.

(C, D) THP1 cells were infected with HSV-1 KOS, ΔICP27 or HSV-1 ΔNLS. The localization of the major NLS is illustrated in panel C. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(E, F) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic (E) and nuclear extracts (F) were isolated at the indicated time points post infection. Western blotting was used to determine levels of phospho- and total TBK1 were determined in the cytoplasmic fractions and levels of phospho-IRF3 and RCC1 in the nuclear fractions.

(G-I) HEK293T cells were (G) transfected with STING, TBK1, IRF3-5D, and ICP27, (H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) or (I) transfected with TRIF, MAVS, STING, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.

Data are presented as means of triplicates +/- st.dev; Symbols for p-values used in the figures: *, 0.01
HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced Type I IFN expression (2016)
Figure 5

Figure 5. ICP27 interacts with the active STING-TBK1 signalsome

(A) Lysates from THP1 cells infected with HSV-1 KOS (MOI 10) were subjected to immunoprecipitation using an anti-STING antibody, and the presence of TBK1, STING, and ICP27 in the precipitates was detected by Western blotting.

(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-pTBK1 (B), or anti-STING (C). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and pTBK1/STING. Scale bar 10 μm.

(D) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF, MAVS, and STING. The precipitates were immunoblotted for TBK1, ICP27, and STING.

(E) THP1 control and STING KO cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27, phospho-TBK1 (pTBK1), and TBK1.

(F) THP1 control and TBK1 KO cells were infected with HSV-1 KOS (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.

(G) THP1 cells were infected with HSV-1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.

(H) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. Phospho-TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and total TBK1.

(I) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and STING (either WT or S366A). The precipitates were immunoblotted for TBK1, ICP27, and STING.
HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced Type I IFN expression (2016)
Figure 6

Figure 6. ICP27 from the simplexvirus genera of alpha-herpesviruses inhibit IFN production in a manner dependent on the RGG box

(A, B) Alignment of the ICP27 homologs of the human herpesviruses (A), and a series of alpha-herpesviruses (B). The diagram to the right illustrates the degree of similarity with HSV-1 ICP27 and the distribution within the molecules.

(C) THP1 cells were infected with HSV-1 (KOS), HSV-2 (HG52), HSV-1 ΔICP27, or HSV-1 ΔICP27 rescued with HSV-2 ICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(D) RAW264.7 cells were infected with HSV-1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

(E) Alignment of the RGG box of in ICP27 proteins of the simplexvirus genera of alpha-herpesviruses.

(F) THP1 cells were infected with HSV-1 (KOS), ΔRGG, or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. ICP27 expression in the infected cells was determined by Western blotting.

(G) THP1 cells were infected with HSV-1 KOS or HSV-1 ΔRGG (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.

Data in panel C, D, and F are presented as means of triplicates +/- st.dev; Symbols for p-values used in the figures: *, 0.01
Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition (2019)
Figure 1

Fig 1. PKM2 interacts with active eNOS.

A. Volcano plot highlighting proteins significantly enriched (open circles, FDR<0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n=3 independent cell batches.

B. Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni).

C. The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls).

D. Consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n=6-19 independent cell batches (1-way ANOVA and Newman-Keuls).

E&F. Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90 and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n=4-8 independent cell batches (1-way ANOVA and Newman-Keuls).

Data information: Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.