EMBO/soda-nel huggingface dataset:
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 1

Figure 1. Upregulation of OTUB1 in astrocytes in MS lesions

(A, B) Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 µm.
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 2

Figure 2. Characterization of astrocyte-specific OTUB1 knockout mice

(A) WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.

(B) Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.

(C) Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 µm. All photographs are representative of three mice per group.

(D) Absolute numbers of CD45+ cells in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (n = 5 for both groups, mean + SEM).

(E) Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry (n = 5 for both groups, mean + SEM).

(F) In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC).

(G) Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488).

(H) GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC).

Data information: (F-H) Original magnification ×400; the inserts show high magnification (×1,200) of the cells marked by an arrow in the respective figures. Scale bars correspond to 50 µm and data are representative of three mice per group.
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 3

Figure 3. Upregulation of OTUB1 in astrocytes limits EAE severity

(A) Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk).

(B) Inflammatory infiltrates in the spinal cord (encircled by a dotted line) of an OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mouse at days 15 and 22 p.i.

(C) At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk).

(D) At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse.

(E) EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35-55 peptide immunization with pertussis toxin. Graph represents data pooled from 4 experiments with 7-8 mice per group and shows the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.

(F) EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35-55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.

Data information: (A-D) All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining.
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 4

Figure 4. Increased leukocyte infiltration and proinflammatory gene transcription in the spinal cord of GFAP-Cre OTUB1fl/fl mice during EAE

(A) Percentages of CD4+ and CD8+ T cells infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. Representative dot plots are shown.

(B) Absolute number of infiltrating leukocytes in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results. Data show mean + SEM. Two-tailed Student's t test was used, * p < 0.05.

(C) Infiltrating CD4+ T cells were further analyzed by flow cytometry for their ability to produce GM-CSF, IFN-γ, and IL-17 at day 15 and 22 p.i., respectively. Representative dot plots are shown.

(D) Absolute number of infiltrating GM-CSF-, IFN-γ-, and IL-17-producing CD4+ T cells at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results (mean + SEM). Two-tailed Student's t test was used, * p < 0.05.

(E) Relative expression of GM-CSF, IFN-γ, IL-17, TNF, IL-6, CXCL1, CXCL10, CXCL11, CCL2 and NOS2 mRNA in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 and 22 p.i. was determined by quantitative real-time PCR. Data are presented as the relative increase over unimmunized control mice (n = 3 for all groups). Data show mean + SEM. Two-tailed Student's t test was used, * p < 0.05.

(F) Astrocytes were isolated from unimmunized and diseased OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 p.i., respectively. Relative expression of CXCL10, CXCL11, CCL2, and NOS2 mRNA was determined by quantitative real-time PCR. Data are presented as relative increase of genes at day 15 p.i. over gene expression of astrocytes from unimmunized control mice (n = 3 for all groups). Data show mean + SEM. Statistical analysis performed using two-tailed Student's t test; * p < 0.05.

(G) Primary astrocytes isolated from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were left untreated or stimulated with IFN-γ (10 ng/ml), IL-17 (50 ng/ml), and TNF (10 ng/ml), respectively, for 16 h. mRNA levels of CXCL10, CXCL11, CCL2, and NOS2 were detected by quantitative real-time PCR. Data are shown as relative increase over untreated controls (mean + SEM). Two-tailed Student's t test was used, * p < 0.05.
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 5

Figure 5. OTUB1 inhibits IFN-γ-activated JAK2-STAT1 signaling pathway by manipulating SOCS1

(A) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times and analyzed by western blot with indicated antibodies.

(B) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times. Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies.

(C) Astrocytes were selectively isolated by MACS from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at days 0 and 15 p.i., respectively. Relative expression of STAT1 mRNA was determined by quantitative real-time PCR. Data show the relative increase of STAT1 mRNA over that of unimmunized control mice (n = 3 for all groups) (mean + SEM). Two-tailed Student's t test was used, * p < 0.05.

(D) Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were stimulated with IFN-γ (10 ng/ml) for indicated times. mRNA was isolated and analyzed by quantitative real-time PCR for SOCS1. Data show the relative increase over untreated controls (mean + SEM). Statistical analysis performed using two-tailed Student's t test; * p < 0.05.

(E) Primary astrocytes from wild type mice were stimulated with IFN-γ (10 ng/ml) for indicated times. Proteins were immunoprecipitated from whole cell lysates with anti-OTUB1 antibody and analyzed by WB with antibodies against JAK1, JAK2, SOCS1, and OTUB1.

(F) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were left untreated or treated with IFN-γ (10 ng/ml) for 30 min. Proteins were immunoprecipitated with anti-SOCS1 antibody and analyzed by WB for SOCS1, OTUB1, and K48 Ubiquitin.

(G) Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC, K48 ubiquitin-HA, and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were left untreated or stimulated with IFN-γ (10 ng/ml) for 30 min before lysation. Proteins were immunoprecipitated with anti-MYC antibody and analyzed by WB for MYC, GFP, and HA.
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 6

Figure 6. OTUB1 inhibits IFN-γ-induced proinflammatory gene expression in astrocytes by stabilizing SOCS1

(A) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Twenty-four hours after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups). Data show mean + SEM. Statistical analysis performed using two-tailed Student's t test; * p < 0.05.

(B) Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids or cotransfected with SOCS1-MYC + OTUB1-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups) (mean + SEM). Two-tailed Student's t test was used, * p < 0.05.

(C) Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (left panel). The right panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 4 for both groups). Data represent mean + SEM. Two-tailed Student's t test was used, * p < 0.05.

(D) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were stimulated with IFN-γ (10 ng/ml) for four hours. Thereafter, cells were treated with CHX (100 μg/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.

(E) Primary astrocytes from OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Five hours later, astrocytes were subsequently transfected with different siRNAs or left untreated. Twenty-four hours after siRNA transfection, protein was isolated and analyzed by WB for SOCS1 expression.

(F) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with nonsense siRNA and SOCS1 siRNA-2. Twenty-four hours after transfection, cells were stimulated with IFN-γ (10 μg/ml) for 16 hours before mRNA isolation. Quantitative real-time PCR was performed to detect mRNA levels of CXCL10, CCL2, and NOS2 (n = 4 for all groups). Data show mean + SEM. Statistical analysis performed using two-tailed Student's t test; * p < 0.05.
OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes (2019)
Figure 7

Figure 7. OTUB1 inhibits IFN-β-induced STAT1 activation and gene transcription

(A) Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-β (10 ng/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.

(B-F) Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were treated with IFN-β (10 ng/ml) for 16 hours. Quantitative real-time PCR was performed to detect OTUB1 (B), CXCL10 (C), CXCL11 (D), CCL2 (E), and NOS2 (F) mRNA levels. Data show mean + SEM. Two-tailed Student's t test was used, * p < 0.05.

Expanded View Figure Legends
Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1 (2021)
Figure 1

Figure 1. Ams1 and Ape4 are Nbr1 interactors and are cargos of the NVT pathway.

(A) Identification of Ams1 and Ape4 as Nbr1 interactors. Nbr1-associated proteins were identified by affinity purification coupled with mass spectrometry (AP-MS) analysis using YFP-FLAG-His6-tagged Nbr1 as bait.

(B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB).

(D-E) Subcellular localization of Ams1 (D) and Ape4 (E). Wild-type (WT), nbr1Δ, sst4Δ, and atg1Δ cells were grown to mid-log phase in EMM medium and examined by live cell imaging. In (D), Ams1 was endogenously tagged at its C-terminus with mCherry. In (E), Ape4 was endogenously tagged at its C-terminus with GFP and in the same cells GFP-binding-protein (GBP) fused 3xUb was expressed under the P41nmt1 promoter. Cpy1 is a vacuole lumen marker. Scale bar, 3 μm.
Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1 (2021)
Figure 2

Figure 2. Ams1 and Ape4 do not rely on other cargos for vacuolar targeting and do not interact with other cargos.

Localization of Ams1 in WT, lap2Δ, ape2Δ, and ape4Δ cells. Scale bar, 3 μm.

Localization of Ape4 in WT, lap2Δ, ape2Δ, and ams1Δ cells. Scale bar, 3 μm.

Ape2 but not Ams1 or Ape4 co-immunoprecipitated with Lap2. Endogenously TAP-tagged Lap2 was immunoprecipitated. Ams1 was endogenously tagged with a 13Myc tag.

Nbr1 but not Lap2 or Ape2 co-immunoprecipitated with Ams1. Endogenously mCherry-tagged Ams1 was immunoprecipitated. Nbr1 was endogenously tagged with a TAP tag.

Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.
Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1 (2021)
Figure 3

Figure 3. ZZ domains in Nbr1 are cargo recognition modules.

Truncation analysis revealed the regions of Nbr1 involved in its interactions with Ams1, Ape4, Lap2, and Ape2. Schematic at the top left shows the domain organization of Nbr1. Pil1 co-tethering assay data are shown in panel B and Appendix Figure S2.

The ZZ1 fragment of Nbr1 interacted with Ams1 and Ape4 in the Pil1 co-tethering assay. In this assay, a bait protein is fused to Pil1, which forms filamentary structures, and a prey protein that can interact with the bait exhibits co-localization on the same structures. Scale bar, 3 μm.

The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ams1.

The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ape4.

Ape4 competes with Ams1 for Nbr1 binding. GST-tagged ZZ1-L1 fragment of Nbr1 was bound to glutathione beads and incubated with FLAG2-His6 (FFH)-tagged Ams1 proteins and different amount of FFH-tagged Ape4. GST-tagged Nbr1 was purified from E. coli. FFH-tagged cargo proteins were purified from S. pombe. Left, SDS-PAGE showing the purify and amounts of the proteins used as input; Right, immunoblotting analysis of the proteins retained on the glutathione beads after washing.

Overexpressing of Ape4 inhibited vacuolar targeting of Ams1. FFH-tagged Ape4 was expressed under the strong Pnmt1 promoter. Scale bar, 3 μm.

The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1. Scale bar, 3 μm.

The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4. Scale bar, 3 μm.

The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2. Scale bar, 3 μm.

The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2. Scale bar, 3 μm.
Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1 (2021)
Figure 6

Figure 6. Mutational analysis of Nbr1-ZZ1 residues involved in cargo recognition

The effects of Nbr1-ZZ1 mutations on its Ams1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Mutations were introduced into the bait protein Pil1-mCherry-ZZ1-ZZ2. Scale bar, 3 μm.

Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.

The effects of Nbr1-ZZ1 mutations on its ability to mediate the vacuolar targeting of Ams1. Mutations were introduced into Nbr1-Δ(ZZ2-ZZ3), which was expressed in the nbr1Δ mutant background. Scale bar, 3 μm.

The effects of Nbr1-ZZ1 mutations on its Ape4-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Mutations were introduced into the bait protein Pil1-mCherry-ZZ1-ZZ2. Scale bar, 3 μm.

SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.

The effects of Nbr1-ZZ1 mutations on its ability to mediate the vacuolar targeting of Ape4. Mutations were introduced into Nbr1-Δ(ZZ2-ZZ3), which was expressed in the nbr1Δ mutant background. Scale bar, 3 μm.
Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1 (2021)
Figure 7

Figure 7. Mutational analysis of cargo residues involved in Nbr1 binding.

The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.

The effects of individually mutating the first two residues of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Pmt3-Ape4 fusions were used to allow alanine substitutions to be introduced at M1 and Q2 positions. Scale bar, 3 μm.

The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.

The effect of preserving the M1 residue of Ams1 on its Nbr1-binding ability. Scale bar, 3 μm.
Glycolytic Pfkp Acts as a Lin41 Protein Kinase to promote endodermal differentiation of Embryonic Stem Cells (2023)
Figure 1

Figure 1. Stat3-dependent regulation of glycolytic enzyme expression in murine ESCs

(A) Expression of all rate-limiting glycolytic isoenzymes in R1 cells after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA (sh-ctrl), as measured by Western blot. β-actin was employed as a loading control throughout.

(B-I) Expression of the pluripotency genes c-Myc, Oct-4, Nanog, Klf4 and Sox-2 in R1 cells after knockdown of Hk2 (B), Pfkl (C), Hk1 (D), Hk3 (E), Pfkm (F), Pkm2 (G), Pklr (H) and Pfkp (I) glycolytic enzymes using two or three independent shRNAs, as measured by Western blot.

Data information: The data were repeated three times and shown as the mean ± SD of three independent experiments. One asterisk and two asterisks indicate p < 0.05 and p < 0.01, respectively.
Glycolytic Pfkp Acts as a Lin41 Protein Kinase to promote endodermal differentiation of Embryonic Stem Cells (2023)
Figure 2

Figure 2. Pfkp is transcriptionally repressed by Stat3 in murine ESCs

(A-C) Expression of Pfkp mRNA levels (top panels) and Stat3 and Pfkp protein expression (bottom panels) after knockdown of Stat3 using three independent shRNAs in either R1 cells (A) or E14 cells (B) or after overexpression of Stat3 in R1 cells (C), as measured by qPCR and Western blot, respectively. β-actin was employed as a loading control throughout.

(D) The levels of Pfkp pre-mRNA compared in sh-ctrl versus sh-Stat3 knockdown R1 cells, as measured by qPCR against the indicated Pfkp introns.

(E, F) Schematic showing the location of three Stat3 binding motifs in the Pfkp gene (E) with binding to Stat3 in R1 cells measured using ChIP assays against Stat3, p-Stat3 (Y705) or isotype-matched immunoglobulin G (IgG) (F), as measured by PCR, and images were representative of three independent experiments. Nanog promoter is shown as a positive control.

(G) Transcriptional activity of the indicated pGL3-based Pfkp promoter or mutant constructs expressed in HEK293T cells with or without overexpression of Stat3, as measured by luciferase reporter assay (top panel). Western blotting was used to verify Stat3 expression (bottom panel).

Data information: The data were repeated three times and shown as the mean ± SD of three independent experiments. One asterisk and two asterisks indicate p < 0.05 and p < 0.01, respectively.