Journal: Immunity

Article Title: The IRF4 gene regulatory module functions as a read-write integrator to dynamically coordinate T helper cell fate

doi: 10.1016/j.immuni.2017.09.001

Figure Lengend Snippet: Irf4 targets distinct DNA motifs to control Tfh and Teff gene programs. ATAC-seq libraries were generated from the same cells in Figure 5. (A) Five patterns of differentially ChARs, A1-5, are shown as box plots (median ±%75th for the box and ±%25th for the whiskers) plotted as a function of averaged Z-scored values. The number of regions within each cluster are shown. (B) Peak tracks at the Bcl6 and Prdm1 loci from the indicated groups; CD4+ naïve and activated are from (GEO: GSE37074). ChARs from a given cluster are highlighted with a downward facing arrowhead and the parental cluster. Peaks with no arrows are not differentially accessible. (C) Heat map depicting the enrichment ratio (Log2) of given Irf4 DNA binding motifs (x-axis) in clusters A1-5 (y-axis); inset text represents the enrichment p-value (Fisher’s Exact Test). (D) Binding saturation curves of Irf4 to the AICE or ISRE motifs. Binding reactions using the AICE probe derived from Bcl11b were carried out in the presence of a constant amount of BATF and JunB containing nuclear extracts. Irf4 containing nuclear extract was increased in 2-fold increments as indicated. The ISRE probe from Prdm1 was used in binding reactions with Irf4 containing nuclear extracts as for the AICE reaction. Nuclear extracts containing a DNA-binding deficient Irf4 (R98A, C99A) were used at the highest concentration of the wild type Irf4 saturation curve as a specificity control. Arrows indicate complexes; AP-1 (blue), Irf4/AP-1 (red), and Irf4 homodimer (green); asterisk indicates non-specific binding. Densitometry analysis is shown to the right of the gel. Representative of two experiments performed. E) Heat map depicting the enrichment ratio of gene members from RNA clusters R1-6 (x-axis) in ChARs A1-5 (y-axis); inset text represents the enrichment p-value (Fisher’s Exact Test). See also Figure S7 and Tables S1–7.

Article Snippet: Plasmid: pMIG backbone expressing JunB , Addgene , Plasmid #40349.

Techniques: Control, Generated, Binding Assay, Derivative Assay, Concentration Assay

Journal: Immunity

Article Title: The IRF4 gene regulatory module functions as a read-write integrator to dynamically coordinate T helper cell fate

doi: 10.1016/j.immuni.2017.09.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pMIG backbone expressing JunB , Addgene , Plasmid #40349.

Techniques: Purification, Control, Recombinant, Transfection, Sensitive Assay, RNA HS Assay, Plasmid Preparation, Expressing, Mutagenesis, Software

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: Primer sets used for PCR and targeted sequences for knockdown studies

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Knockdown, Cloning

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: TF expression, activation, and the impact of PAX5 on HCK transcription in MYD88-mutated lymphoma cells. (A) Western blot studies depicting protein expression levels of PAX5, STAT3, NF-kB (NF-κB-p65), and AP-1 complex members (JunB, c-Jun, JunD) predicted by TF promoter-binding assay and PROMO analysis as HCK promoter binding TFs in MYD88-mutated WM and ABC-DLBCL cell lines (BCWM.1, MWCL-1, TMD-8, HBL-1, OCI-Ly3, and SU-DHL-2) and MYD88 wild-type B-cell lymphoma (OCI-Ly7, OCI-Ly19, Ramos) and myeloma cells (RPMI-8226, MM.1S). The HCK protein expression levels and the phosphorylation levels of mutated MYD88–directed TFs STAT3, NF-κB-p65, and AP-1 complex members (JunB, c-Jun) were also detected. GAPDH protein expression was used to demonstrate uniform protein loading. (B) The regulation of HCK transcription by PAX5 was assessed by lentiviral knockdown of PAX5 with 2 distinct shRNAs in MYD88-mutated BCWM.1 and TMD-8 cells and compared with scrambled control vector. Quantitative RT-PCR was performed after day 5 of lentiviral transduction. HCK protein levels and knockdown efficiencies for PAX5 were analyzed by western blot at the same time as the sample collection for HCK mRNA quantification. GAPDH was used for loading control. (C) The regulation of PAX5 by mutated MYD88 was assessed by lentiviral-mediated knockdown of MYD88 in MYD88-mutated BCWM.1 and TMD-8 cells using 2 distinct shRNAs and compared with scrambled control vector. Protein levels of PAX5 are shown, and GADPH served as a protein loading control. (D) Transcriptome analysis depicting PAX5 transcript levels in CD19-selected bone marrow LPCs from MYD88-mutated WM patients, and MYD88 wild-type WM patients; peripheral CD19-selected B cells and CD19- and CD27-selected memory B cells from healthy donors; and CD138-selected bone marrow plasma cells from healthy donors. ***P < .001.

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Western Blot, Binding Assay, Phospho-proteomics, Knockdown, Control, Plasmid Preparation, Quantitative RT-PCR, Transduction, Clinical Proteomics

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: ChIP studies assessing STAT3, NF-kB, and AP-1 TF binding to the HCK promoter. The fold enrichments of HCK promoter-specific sequence assessed by quantitative PCR following ChIP with ChIP grade antibodies to STAT3, NF-kB-p65, JunB, and c-Jun in MYD88-mutated WM (BCWM.1, MWCL-1) and ABC-DLBCL (TMD-8, HBL-1, OCI-Ly3) cells, and MYD88 wild-type lymphoma cells (OCI-Ly7, OCI-Ly19). Antibody to GAPDH was used as control antibody.

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, ChIP-chip, Control

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: The regulation of JunB by TLR/MYD88 signaling and the impact of JunB on HCK transcription. The phosphorylation of JunB (Ser79) and c-Jun (Ser63) was assessed following TLR4 (by LPS-EB) and TLR9 (by ODN-2006) stimulation (A) as well as the lentiviral cells (C). HCK overexpression of MYD88 L265P mutant vs MYD88 WT (B) in both MYD88-mutated BCWM.1 cells and MYD88 wild-type Ramos cells. The phosphorylation of JunB (Ser79) and c-Jun (Ser63) was determined following MYD88 knockdown in MYD88-mutated BCWM.1 protein levels detected following lentiviral mediated knockdown of JunB in MYD88-mutated BCWM.1 and TMD-8 cells using 2 distinct shRNAs and compared with scrambled control vector (D). Protein levels of MYD88, JunB, c-Jun, and GADPH served as protein expression, knockdown efficiency, and loading controls.

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Phospho-proteomics, Over Expression, Mutagenesis, Knockdown, Control, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Allelic effects on KLHL17 expression underlie a pancreatic cancer genome-wide association signal at chr1p36.33

doi: 10.1038/s41467-025-59109-2

Figure Lengend Snippet: Predicted TFs with allelic binding preferences for rs13303327 and rs13303160

Article Snippet: Recombinant ELF1 (TP760629), ELF2 (TP760288), ELF3 (TP300631), ELF4 (TP761826), JUNB (TP303595), JUND (TP316958 4), c-FOS (TP760257), FOS1L (TP302104), FOSB (TP762032), FOS2L (TP760114) proteins were purchased from Origene (Rockville, MD). c-JUN was purchased from Abcam (Waltham, MA) (ab84134).

Techniques: Binding Assay, Significance Assay

Journal: Nature Communications

Article Title: Allelic effects on KLHL17 expression underlie a pancreatic cancer genome-wide association signal at chr1p36.33

doi: 10.1038/s41467-025-59109-2

Figure Lengend Snippet: a in silico Transcription factor binding predictions; the risk (G) allele disrupts the motif. b Representative EMSA using TPA-stimulated nuclear HeLa extract and fluorescently labeled oligonucleotide ( n = 3 independent experiments). Arrow indicates the allele-preferential binding. c Luciferase reporter assay using DMSO and TPA stimulation and the rs13303160 sequence as an enhancer in the PANC-1 cell line; luciferase activity reported relative to the Empty Vector (EV, gray bars). Pink bars denote the risk allele, and blue bars represent the protective allele. Both alleles were tested in the forward (fwd) and reverse (rev) orientations. Unpaired, two-tailed t tests were performed on the relative luciferase activity of the A/G ratio compared to A/A; n = 6 biological replicates. d Representative EMSAs with increasing amounts of recombinant Fos proteins (from left to right: c-Fos ( n = 3 independent experiments); FosB ( n = 2); Fos1L ( n = 1); Fos2L ( n = 1)). e Representative EMSAs with increasing amounts of recombinant Jun proteins (from left to right: c-Jun ( n = 2 independent experiments), JunB ( n = 2), JunD ( n = 2)). Arrow indicates the allele-specific binding. f , g Representative supershift EMSA with antibodies against JunB and JunD ( n = 3 independent experiments each), respectively, using both TPA-stimulated nuclear lysate and recombinant protein; Arrows denote the shift in the bands. h ChIP-qPCR in SW1990 PDAC cells for JunB (denoted in blue, IgG in gray) ( n = 6 biological replicates) and JunD (denoted in blue, IgG in gray) ( n = 4 biological replicates) using 3 primer sets (PS) surrounding the SNP. Positive control (PC) is from a JunB ChIP-seq performed in the CFPAC1 PDAC cell line . Negative control (NC) is from a quiescent region on chr1p36.33; i TaqMan genotyping assay for rs13303160 using immunoprecipitated DNA from the ChIP. The ratio of A to G was determined relative to the quantity of A and G alleles in the input DNA (gray) ( n = 6 biological replicates for JunB; n = 5 biological replicates for JunD, blue bars). Red font denotes the risk allele in ( b – g ). For all graphs, error bars represent the SEM. Unpaired two-tailed t tests were performed. Source data are provided as a Source Data file.

Article Snippet: Recombinant ELF1 (TP760629), ELF2 (TP760288), ELF3 (TP300631), ELF4 (TP761826), JUNB (TP303595), JUND (TP316958 4), c-FOS (TP760257), FOS1L (TP302104), FOSB (TP762032), FOS2L (TP760114) proteins were purchased from Origene (Rockville, MD). c-JUN was purchased from Abcam (Waltham, MA) (ab84134).

Techniques: In Silico, Binding Assay, Labeling, Luciferase, Reporter Assay, Sequencing, Activity Assay, Plasmid Preparation, Two Tailed Test, Recombinant, ChIP-qPCR, Positive Control, ChIP-sequencing, Negative Control, Genotyping Assay, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: PKCθ-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

doi: 10.1038/s41419-020-2522-0

Figure Lengend Snippet: a Western blot analysis of control and various time periods of VEGFA (40 ng/ml)-treated HRMVECs for the indicated proteins. b The effect of siControl, siPKCθ and siJunB (100 nM) on VEGFA (40 ng/ml)-induced JunB expression at 2 h and PKCθ phosphorylation at 10 min. The blot was sequentially reprobed for PKCθ, JunB, β-tubulin and α-tubulin levels to show the specificity and efficacy of the siRNA on its target and off target molecules. c Upper panel: Western blot analysis of JunB and β-tubulin levels to show the specificity and efficacy of siControl and siJunB (100 nM) in HRMVECs. Bottom panel: The effect of siControl and siJunB on VEGFA (40 ng/ml)-induced HRMVEC migration. d – f All the conditions were same as in ( c ) except that cells were treated with and without VEGFA (40 ng/ml) and DNA synthesis ( d ), sprouting ( e ) or tube formation ( f ) were measured. The bar graphs represent quantitative analysis of three independent experiments. The values are presented as mean ± SD. * p < 0.01 vs vehicle control or siControl; ** p < 0.01 vs siControl + VEGFA. Scale bars in ( e ) and ( f ) are 50 and 200 μm, respectively.

Article Snippet: Mouse JunB overexpression plasmid (Cat. No. MR336772) was obtained from Origene Technologies Inc. (Rockville, MD).

Techniques: Western Blot, Control, Expressing, Phospho-proteomics, Migration, DNA Synthesis

Journal: Cell Death & Disease

Article Title: PKCθ-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

doi: 10.1038/s41419-020-2522-0

Figure Lengend Snippet: a Western blot analysis of retinal extracts of normoxic and 12 h and 24 h of hypoxic WT mice pups for the indicated proteins. b Western blot analysis of retinal extracts of normoxic and 24 h of hypoxic WT and PKCθ −/− mice pups for JunB levels. The blot was subsequently reprobed for PKCθ and β-tubulin levels. c Left panel: Normoxic or hyperoxic JunB flox/flox :Cdh5-Cre ERT2 mice pups at P10 and P11 were injected with 100 μg tamoxifen intraperitoneally, and normoxic and 24 h of hypoxic (P13) pup retinal extracts were prepared and analyzed by western blotting for phospho and total PKCθ, JunB and β-tubulin levels. Right panel: All the conditions were the same as in the left panel except that eyes from normoxic and 72 h of hypoxic pups were enucleated, fixed, sections were made and co-immunostained for CD31 and JunB. d All the conditions are same as in right ( c ) except that eyes were enucleated from normoxic and 120 h of hypoxic pups, fixed, retinas isolated, stained with isolectin B4, flat mounts were made and retinal vascularization and neovascularization were measured. Retinal vascularization is sown in the first column at 2.5× magnification (scale bar 500 μm) and neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column at 10× magnification (scale bar, 200 μm). e , f Retinal neovascularization ( e ) and avascular area ( f ) were determined as described in “Materials and Methods”. g Upper panel: All the conditions were the same as in left ( c ) except that sections were co-immunostained for CD31 and Ki67. The extreme right column shows 40× magnification of the areas selected by rectangular boxes in the immediate left column images (scale bars in the far left and far right columns are 200 and 50 μm, respectively). Retinal EC proliferation was measured by counting CD31 and Ki67-positive cells from the inner limiting membrane to the extended region in each section ( n = 6 eyes, 3 sections/eye). Bottom left panel: Quantitative analysis of CD31 and Ki67-positive cells. Bottom right panel: The effect of siControl and siJunB on VEGFA (40 ng/ml)-induced DNA synthesis in MRMVECs. h All the conditions were the same as in ( d ) except that the retinal flat mounts were examined for filopodia at 40× magnification (scale bar, 50 μm). The bar graphs represent quantitative analysis of three blots or 7 retinas. The values are presented as mean ± SD. * p < 0.01 vs normoxia or WT normoxia or siControl; ** p < 0.01 vs WT hypoxia, siControl + hypoxia or siControl + VEGFA.

Article Snippet: Mouse JunB overexpression plasmid (Cat. No. MR336772) was obtained from Origene Technologies Inc. (Rockville, MD).

Techniques: Western Blot, Injection, Isolation, Staining, Membrane, DNA Synthesis

Journal: Cell Death & Disease

Article Title: PKCθ-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

doi: 10.1038/s41419-020-2522-0

Figure Lengend Snippet: a Western blot analysis of control and the indicated time periods of VEGFA (40 ng/ml)-treated HRMVECs for VEGFR3 and β-tubulin levels. b , c The effect of siControl, siPKCθ and siJunB (100 nM) on VEGFA (40 ng/ml)-induced (2 h) VEGFR3 levels. The blots were sequentially reprobed for PKCθ and β-tubulin levels or JunB and β-tubulin levels to show the specificity and efficacy of siRNA on its target and off target molecules. d Upper panel: Retinal ECs were isolated from WT and PKCθ −/− mice and tested for the effect of VEGFA on PKCθ phosphorylation and JunB and VEGFR3 expression. Lower panel: Retinal ECs from PKCθ −/− mice were transfected with empty vector or JunB expression vector and two days later cell extracts were prepared and analyzed by western blotting for JunB, VEGFR3 and β-tubulin levels. e Upper panel: western blot analysis of VEGFR2, VEGFR3 and β-tubulin levels to show the specificity and efficacy of siControl and siVEGFR3 (100 nM) in HRMVECs. Bottom panel: The effect of siControl, siVEGFR3 and MAZ51 (5 μM) on VEGFA (40 ng/ml)-induced HRMVEC migration. f – h All the conditions were same as in ( e ) except that cells were treated with and without VEGFA (40 ng/ml) and DNA synthesis ( f ), sprouting ( g ) or tube formation ( h ) were measured. The bar graphs represent quantitative analysis of three independent experiments. The values are presented as mean ± SD. * p < 0.01 vs vehicle control or siControl; ** p < 0.01 vs siControl + VEGFA. Scale bars in ( g ) and ( h ) are 50 and 200 μm, respectively.

Article Snippet: Mouse JunB overexpression plasmid (Cat. No. MR336772) was obtained from Origene Technologies Inc. (Rockville, MD).

Techniques: Western Blot, Control, Isolation, Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Migration, DNA Synthesis

Journal: Cell Death & Disease

Article Title: PKCθ-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

doi: 10.1038/s41419-020-2522-0

Figure Lengend Snippet: a Western blot analysis of retinal extracts of normoxic and the indicated time periods of hypoxic WT mice pups for VEGFR1, VEGFR2, VEGFR3 and β-tubulin levels. b Upper panel: Mice pups were injected intravitreally with 1 μg/0.5 μl/eye of siControl or siVEGFR3 at P11 and P12 and at P13, retinal extracts were prepared and analyzed by western blotting for VEGFR2, VEGFR3 and β-tubulin levels. Bottom panel: All the conditions were the same as in upper panel except that pups were received siRNA at P11, P12 and P13 and at P15, eyes were enucleated, fixed, sections were made and immunostained for CD31 and VEGFR3. c All the conditions are same as in bottom ( b ) except that eyes were enucleated at P17, fixed, retinas were isolated, stained with isolectin B4 and flat mounts were made. Retinal vascularization is shown in the first column at 2.5× magnification (scale bar, 1000 μm). Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column at 10× magnification (scale bar, 200 μm). d , e . Retinal neovascularization ( d ) and avascular area ( e ) were determined as described in “Materials and Methods.” f The effect of siControl and siVEGFR3 on VEGFA (40 ng/ml)-induced DNA synthesis in MRMVECs. g Left panel: All the conditions were the same as in ( c ) except that the retinal flat mounts were examined for filopodia at 40× magnification (scale bar, 50 μm). Right panel: Quantitative analysis of number of filopodia/unit vessel length. h Upper left panel: western blot analysis of normoxic and 24 h of hypoxic retina of WT and PKCθ −/− mice pups for VEGFR3, PKCθ and β-tubulin levels. Bottom left panel: All the conditions were the same as in upper panel except that eyes were enucleated at 3 days of hypoxia (P15), fixed, sections were made and immunostained for CD31 and VEGFR3. Upper and bottom right panels: Quantitative analysis of VEGFR3 levels and CD31 and VEGFR3-positive cells, respectively. i Upper left panel: Normoxic or hyperoxic JunB flox/flox :Cdh5-Cre ERT2 mice pups at P10 and P11 were injected with 100 μg tamoxifen intraperitoneally, and RNA was isolated from normoxic and 24 h of hypoxic (P13) pup retinas and analyzed by RT-PCR for VEGFR3 and α-actin mRNA levels. Upper right panel: All the conditions were the same as in the left panel except that retinal extracts were prepared and analyzed for western blotting for VEGFR3, JunB and β-tubulin levels. Lower panel: All the conditions were the same as in the upper panels except that eyes were enucleated, fixed, sections were made and co-immunostained for CD31 and VEGFR3. The bar graphs represent quantitative analysis of three blots or 7 retinas. The values are presented as mean ± SD. * p < 0.01 vs normoxia or WT + normoxia or siControl + normoxia; ** p < 0.01 vs WT + hypoxia or siControl + hypoxia.

Article Snippet: Mouse JunB overexpression plasmid (Cat. No. MR336772) was obtained from Origene Technologies Inc. (Rockville, MD).

Techniques: Western Blot, Injection, Isolation, Staining, DNA Synthesis, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: PKCθ-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

doi: 10.1038/s41419-020-2522-0

Figure Lengend Snippet: a Upper panel: Equal amount of protein from control and the indicated time periods of VEGFA-treated HRMVECs were analyzed for phospho and total VEGFR1, 2 and 3 levels. Lower panel: The cell extracts were analyzed for VEGFR2 and VEGFR3 complex formation. b HRMVECs were transfected with siControl or siVEGFR2 (100 nM), quiesced, treated with and without VEGFA (40 ng/ml) for 10 min (for pPKCθ) or 120 min (for JunB and VEGFR3) and cell extracts were prepared and analyzed by western blotting for the indicated proteins. c Retinas from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that received siControl or siVEGFR2 (1 μg/0.5 μl/eye) by intravitreal injections at P10 and P11 were isolated, extracts were prepared and analyzed by western blotting for the indicated proteins. d Eyes from normoxic and 72 h (i.e., at P15) of hypoxic WT mice pups that received siControl or siVEGFR2 (1 μg/0.5 μl/eye) by intravitreal injections at P11, P12 and P13 were enucleated, fixed, sections were made and coimmunostained for CD31 and VEGFR2 (left panel) and CD31 and VEGFR3 (right panel). e Eyes from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that were injected intravitreally with vehicle or 0.05 μg/0.5 μl/eye of soluble VEGFR2 at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for the indicated proteins using their specific antibodies. f Eyes from normoxic and 24 h of hypoxic mice pups that were injected intravitreally with siControl or siVEGFR2 (0.5 μg/0.5 μl/eye) at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for VEGFR3 levels using its specific antibodies and the blot was normalized for β-tubulin. g All the conditions were the same as in ( d ) except that sections were coimmunostained for CD31 and Ki67. The bar graph shows quantification of proliferating ECs per section. h All the conditions were the same as in ( d ) except that eyes were enucleated at P17, fixed, retinas were isolated, stained with isolectin B4, flat mounts were made and examined for filopodia at 40× magnification (scale bar, 50 μm). Bar graph shows quantification of the number of filopodia/unit vessel length. i All the conditions were the same as in ( h ) except that the flat mounts were examined for retinal vascularization. Retinal vascularization is shown in the first column at 2.5× magnification (scale bar, 500 μm). Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column at 10× magnification (scale bar, 200 μm). j , k Retinal neovascularization ( j ) and avascular area ( k ) were determined as described in “Materials and Methods.” The values are presented as mean ± SD. * p < 0.01 vs normoxia; ** p < 0.01 vs siControl + hypoxia.

Article Snippet: Mouse JunB overexpression plasmid (Cat. No. MR336772) was obtained from Origene Technologies Inc. (Rockville, MD).

Techniques: Control, Transfection, Western Blot, Isolation, Injection, Staining