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a VSMCs infected with pAd-GFP or pAd-GFP-KLF4 were treated with 20 μg/ml of cycloheximide (CHX) for the indicated times. PFKFB3 protein levels were detected by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs CHX 0 h+pAd-GFP, ### P < 0.005 vs CHX 0 h+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). b <t>eEF1A2</t> mRNA levels were assessed by qRT-PCR. **** P < 0.001 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). c eEF1A2 protein levels in mock- and KLF4-transduced VSMCs were measured by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). d A Venn diagram showing 40 overlapping proteins between KLF4- and eEF1A2-overexpressing VSMCs identified by the TMT-based LC-MS/MS analysis. Two proteins were simultaneously upregulated more than 2-fold by KLF4 and eEF1A2 (upper), and 38 proteins had a 1.2-fold increase (lower). e VSMCs were infected with pAd-GFP or pAd-eEF1A2 with a flag tag. PFKFB3 protein levels were measured by immunoblotting and quantified by normalizing to β-actin ( n = 3 independent experiments, error bars show SEM). f VSMCs were transfected with the indicated constructs. PFKFB3, eEF1A2 and β-actin protein levels were measured by immunoblotting and PFKFB3 protein levels were quantified by normalizing to β-actin. *** P < 0.005 vs si-Con+pAd-GFP, ### P < 0.005 vs si-Con+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). g , h circRNA microarrays were performed in VSMCs treated as indicated. Volcano plots are used for visualizing differentially expressed circRNAs between the two groups (fold change>1.5, P < 0.05, n = 3 independent samples each group). The red blocks indicate differentially expressed circRNAs; gray blocks indicate circRNAs with no difference in their expression ( g ). 31 upregulated circRNAs by KLF4 (fold change>4) were selected and summarized ( h ). i qRT-PCR detected the indicated circRNAs in VSMCs treated as indicated. * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). One-way ANOVA with Tukey’s multiple comparison tests were performed for a – c , f , i . Unpaired Student’s t tests were performed for e .
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IDH mutations activate CEBPα-VDR-RXRα axis through 2HG production. ( A ) RT-qPCR showing gene expressions of VDR and RARA in HL60 IDH1 WT (clone 2: ○; clone 7: ☐) versus HL60 IDH1 R132H (clone 5: ◇; clone 11: △). ( B ) Western blot showing protein levels of VDR, RXRα and RARα in HL60 IDH1 WT (clones 2 and 7) versus HL60 IDH1 R132H (clones 5, 11 and 3) (left) and in HL60 <t>IDH2</t> WT versus HL60 IDH2 R172K (right). ( C ) RT-qPCR showing gene expressions of VDR and RARA in 2HG-treated HL60 (200 µM, 1 week). ( D ) Western blot showing protein levels of VDR RXRα and RARα in 2HG-treated HL60 IDH1 WT (clones 2 and 7) (200 µM, 1 week). ( E ) RT-qPCR showing gene expression of and VDR and RARA in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) and IDH1 R132H (clone 5: ◇, clone 11: △) after CEBPA-KD by shRNA ( F ) Western blot showing protein levels of CEBPα, VDR, RARα and VDR in HL60 IDH1 WT (clones 2 and 7) and IDH1 R132H (clone 5 and 11) after CEBPA-KD by shRNA. ( G ) Western blot showing protein levels of VDR in 2HG-treated HL60 IDH1 WT (clone 2 and 7) with CEBPA-KD by shRNA (200 µM, 1 week). The uncropped western blot figures are presented in . * p < 0.05; ** p < 0.01.
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a VSMCs infected with pAd-GFP or pAd-GFP-KLF4 were treated with 20 μg/ml of cycloheximide (CHX) for the indicated times. PFKFB3 protein levels were detected by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs CHX 0 h+pAd-GFP, ### P < 0.005 vs CHX 0 h+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). b eEF1A2 mRNA levels were assessed by qRT-PCR. **** P < 0.001 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). c eEF1A2 protein levels in mock- and KLF4-transduced VSMCs were measured by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). d A Venn diagram showing 40 overlapping proteins between KLF4- and eEF1A2-overexpressing VSMCs identified by the TMT-based LC-MS/MS analysis. Two proteins were simultaneously upregulated more than 2-fold by KLF4 and eEF1A2 (upper), and 38 proteins had a 1.2-fold increase (lower). e VSMCs were infected with pAd-GFP or pAd-eEF1A2 with a flag tag. PFKFB3 protein levels were measured by immunoblotting and quantified by normalizing to β-actin ( n = 3 independent experiments, error bars show SEM). f VSMCs were transfected with the indicated constructs. PFKFB3, eEF1A2 and β-actin protein levels were measured by immunoblotting and PFKFB3 protein levels were quantified by normalizing to β-actin. *** P < 0.005 vs si-Con+pAd-GFP, ### P < 0.005 vs si-Con+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). g , h circRNA microarrays were performed in VSMCs treated as indicated. Volcano plots are used for visualizing differentially expressed circRNAs between the two groups (fold change>1.5, P < 0.05, n = 3 independent samples each group). The red blocks indicate differentially expressed circRNAs; gray blocks indicate circRNAs with no difference in their expression ( g ). 31 upregulated circRNAs by KLF4 (fold change>4) were selected and summarized ( h ). i qRT-PCR detected the indicated circRNAs in VSMCs treated as indicated. * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). One-way ANOVA with Tukey’s multiple comparison tests were performed for a – c , f , i . Unpaired Student’s t tests were performed for e .

Journal: Communications Biology

Article Title: KLF4-PFKFB3-driven glycolysis is essential for phenotypic switching of vascular smooth muscle cells

doi: 10.1038/s42003-022-04302-y

Figure Lengend Snippet: a VSMCs infected with pAd-GFP or pAd-GFP-KLF4 were treated with 20 μg/ml of cycloheximide (CHX) for the indicated times. PFKFB3 protein levels were detected by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs CHX 0 h+pAd-GFP, ### P < 0.005 vs CHX 0 h+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). b eEF1A2 mRNA levels were assessed by qRT-PCR. **** P < 0.001 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). c eEF1A2 protein levels in mock- and KLF4-transduced VSMCs were measured by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). d A Venn diagram showing 40 overlapping proteins between KLF4- and eEF1A2-overexpressing VSMCs identified by the TMT-based LC-MS/MS analysis. Two proteins were simultaneously upregulated more than 2-fold by KLF4 and eEF1A2 (upper), and 38 proteins had a 1.2-fold increase (lower). e VSMCs were infected with pAd-GFP or pAd-eEF1A2 with a flag tag. PFKFB3 protein levels were measured by immunoblotting and quantified by normalizing to β-actin ( n = 3 independent experiments, error bars show SEM). f VSMCs were transfected with the indicated constructs. PFKFB3, eEF1A2 and β-actin protein levels were measured by immunoblotting and PFKFB3 protein levels were quantified by normalizing to β-actin. *** P < 0.005 vs si-Con+pAd-GFP, ### P < 0.005 vs si-Con+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). g , h circRNA microarrays were performed in VSMCs treated as indicated. Volcano plots are used for visualizing differentially expressed circRNAs between the two groups (fold change>1.5, P < 0.05, n = 3 independent samples each group). The red blocks indicate differentially expressed circRNAs; gray blocks indicate circRNAs with no difference in their expression ( g ). 31 upregulated circRNAs by KLF4 (fold change>4) were selected and summarized ( h ). i qRT-PCR detected the indicated circRNAs in VSMCs treated as indicated. * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001 vs pAd-GFP ( n = 3 independent experiments, error bars show SEM). One-way ANOVA with Tukey’s multiple comparison tests were performed for a – c , f , i . Unpaired Student’s t tests were performed for e .

Article Snippet: Immunofluorescence staining of mouse aortic root paraffin sections was performed with primary antibodies anti-CD11c (1:100 dilution, ab11029, Abcam), anti-KLF4 (1:100 dilution, ab215036, Abcam), anti-PFKFB3 (1:100 dilution, ab181861, Abcam), anti-eEF1A2 (1:100 dilution, 16091-1-AP, Proteintech) and anti-SMα-actin (1:200 dilution, ab32575 or ab240654, Abcam).

Techniques: Infection, Western Blot, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, FLAG-tag, Transfection, Construct, Expressing

a VSMCs were transfected with the indicated constructs. PFKFB3, flag and β-actin protein levels were measured by immunoblotting. #1 to 15 represent constructs of circZFAT, circCTDP1, circCUX1, circRPS6KA1, circTM4SF-TCTEX1D2, circTSNARE1, circDDX42, circTNRC6B, circZBTB46, circADAMTS17, circTMEM209, circCNDP2, circTEX2, circFAM53B-1, and circFAM53B-2, respectively. b Schematic illustration showing two targeted siRNAs. si-circRNA targets the back-splice junction of circCTDP1 or circZFAT, and si-both targets both the linear and circular transcripts. c qRT-PCR detected the indicated circRNAs and mRNAs in VSMCs transfected with the two siRNAs shown in b . *** P < 0.005 and **** P < 0.001 vs si-Con ( n = 3 independent experiments, error bars show SEM). d – f VSMCs were transfected with the indicated constructs. PFKFB3 protein levels were measured by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs si-Con+pAd-GFP, ## P < 0.01 and ### P < 0.005 vs si-Con+pAd-GFP-KLF4, $$$ P < 0.005 vs si-circCTDP1+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). g RNA immunoprecipitation (RIP) was performed with anti-IgG or anti-eEF1A2 antibody in lysates of VSMCs infected with pAd-GFP-KLF4, and then the immunoprecipitates were used to detect circCTDP1 by qRT-PCR. **** P < 0.001 vs IgG ( n = 3 independent experiments, error bars show SEM). h , i RNA pull-down assay was performed in VSMCs infected with pAd-GFP or pAd-GFP - KLF4. Cell lysates were pulled down with probe against circCTDP1. qRT-PCR detected circCTDP1 ( h ). **** P < 0.001 vs pAd-GFP ( n = 4 independent experiments, error bars show SEM). Western blotting detected eEF1A2 ( i ). j Representative immunofluorescent staining of eEF1A2 (green), circCTDP1 (red) and DAPI (blue) in VSMCs (bars = 50 μm). White arrow showed the co-localization between eEF1A2 and circCTDP1. Unpaired Student’s t tests were performed for c and g . One-way ANOVA with Tukey’s multiple comparison tests were performed for d – f , h .

Journal: Communications Biology

Article Title: KLF4-PFKFB3-driven glycolysis is essential for phenotypic switching of vascular smooth muscle cells

doi: 10.1038/s42003-022-04302-y

Figure Lengend Snippet: a VSMCs were transfected with the indicated constructs. PFKFB3, flag and β-actin protein levels were measured by immunoblotting. #1 to 15 represent constructs of circZFAT, circCTDP1, circCUX1, circRPS6KA1, circTM4SF-TCTEX1D2, circTSNARE1, circDDX42, circTNRC6B, circZBTB46, circADAMTS17, circTMEM209, circCNDP2, circTEX2, circFAM53B-1, and circFAM53B-2, respectively. b Schematic illustration showing two targeted siRNAs. si-circRNA targets the back-splice junction of circCTDP1 or circZFAT, and si-both targets both the linear and circular transcripts. c qRT-PCR detected the indicated circRNAs and mRNAs in VSMCs transfected with the two siRNAs shown in b . *** P < 0.005 and **** P < 0.001 vs si-Con ( n = 3 independent experiments, error bars show SEM). d – f VSMCs were transfected with the indicated constructs. PFKFB3 protein levels were measured by immunoblotting and quantified by normalizing to β-actin. *** P < 0.005 vs si-Con+pAd-GFP, ## P < 0.01 and ### P < 0.005 vs si-Con+pAd-GFP-KLF4, $$$ P < 0.005 vs si-circCTDP1+pAd-GFP-KLF4 ( n = 3 independent experiments, error bars show SEM). g RNA immunoprecipitation (RIP) was performed with anti-IgG or anti-eEF1A2 antibody in lysates of VSMCs infected with pAd-GFP-KLF4, and then the immunoprecipitates were used to detect circCTDP1 by qRT-PCR. **** P < 0.001 vs IgG ( n = 3 independent experiments, error bars show SEM). h , i RNA pull-down assay was performed in VSMCs infected with pAd-GFP or pAd-GFP - KLF4. Cell lysates were pulled down with probe against circCTDP1. qRT-PCR detected circCTDP1 ( h ). **** P < 0.001 vs pAd-GFP ( n = 4 independent experiments, error bars show SEM). Western blotting detected eEF1A2 ( i ). j Representative immunofluorescent staining of eEF1A2 (green), circCTDP1 (red) and DAPI (blue) in VSMCs (bars = 50 μm). White arrow showed the co-localization between eEF1A2 and circCTDP1. Unpaired Student’s t tests were performed for c and g . One-way ANOVA with Tukey’s multiple comparison tests were performed for d – f , h .

Article Snippet: Immunofluorescence staining of mouse aortic root paraffin sections was performed with primary antibodies anti-CD11c (1:100 dilution, ab11029, Abcam), anti-KLF4 (1:100 dilution, ab215036, Abcam), anti-PFKFB3 (1:100 dilution, ab181861, Abcam), anti-eEF1A2 (1:100 dilution, 16091-1-AP, Proteintech) and anti-SMα-actin (1:200 dilution, ab32575 or ab240654, Abcam).

Techniques: Transfection, Construct, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Infection, Pull Down Assay, Staining

IDH mutations activate CEBPα-VDR-RXRα axis through 2HG production. ( A ) RT-qPCR showing gene expressions of VDR and RARA in HL60 IDH1 WT (clone 2: ○; clone 7: ☐) versus HL60 IDH1 R132H (clone 5: ◇; clone 11: △). ( B ) Western blot showing protein levels of VDR, RXRα and RARα in HL60 IDH1 WT (clones 2 and 7) versus HL60 IDH1 R132H (clones 5, 11 and 3) (left) and in HL60 IDH2 WT versus HL60 IDH2 R172K (right). ( C ) RT-qPCR showing gene expressions of VDR and RARA in 2HG-treated HL60 (200 µM, 1 week). ( D ) Western blot showing protein levels of VDR RXRα and RARα in 2HG-treated HL60 IDH1 WT (clones 2 and 7) (200 µM, 1 week). ( E ) RT-qPCR showing gene expression of and VDR and RARA in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) and IDH1 R132H (clone 5: ◇, clone 11: △) after CEBPA-KD by shRNA ( F ) Western blot showing protein levels of CEBPα, VDR, RARα and VDR in HL60 IDH1 WT (clones 2 and 7) and IDH1 R132H (clone 5 and 11) after CEBPA-KD by shRNA. ( G ) Western blot showing protein levels of VDR in 2HG-treated HL60 IDH1 WT (clone 2 and 7) with CEBPA-KD by shRNA (200 µM, 1 week). The uncropped western blot figures are presented in . * p < 0.05; ** p < 0.01.

Journal: Cancers

Article Title: Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

doi: 10.3390/cancers13205243

Figure Lengend Snippet: IDH mutations activate CEBPα-VDR-RXRα axis through 2HG production. ( A ) RT-qPCR showing gene expressions of VDR and RARA in HL60 IDH1 WT (clone 2: ○; clone 7: ☐) versus HL60 IDH1 R132H (clone 5: ◇; clone 11: △). ( B ) Western blot showing protein levels of VDR, RXRα and RARα in HL60 IDH1 WT (clones 2 and 7) versus HL60 IDH1 R132H (clones 5, 11 and 3) (left) and in HL60 IDH2 WT versus HL60 IDH2 R172K (right). ( C ) RT-qPCR showing gene expressions of VDR and RARA in 2HG-treated HL60 (200 µM, 1 week). ( D ) Western blot showing protein levels of VDR RXRα and RARα in 2HG-treated HL60 IDH1 WT (clones 2 and 7) (200 µM, 1 week). ( E ) RT-qPCR showing gene expression of and VDR and RARA in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) and IDH1 R132H (clone 5: ◇, clone 11: △) after CEBPA-KD by shRNA ( F ) Western blot showing protein levels of CEBPα, VDR, RARα and VDR in HL60 IDH1 WT (clones 2 and 7) and IDH1 R132H (clone 5 and 11) after CEBPA-KD by shRNA. ( G ) Western blot showing protein levels of VDR in 2HG-treated HL60 IDH1 WT (clone 2 and 7) with CEBPA-KD by shRNA (200 µM, 1 week). The uncropped western blot figures are presented in . * p < 0.05; ** p < 0.01.

Article Snippet: For the generation of IDH2 WT or IDH2 R72K HL60, the following pSLIK vectors were used: pSLIK-IDH2-FLAG (Addgene plasmid #66806); pSLIK-IDH2-R172K-FLAG (Addgene plasmid #66807).

Techniques: Quantitative RT-PCR, Western Blot, Clone Assay, Expressing, shRNA

Combine treatment with VD and ATRA produces a synergistic induction of differentiation in a CEBPα-dependent manner. ( A ) HeatMap of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 WT versus HL60 IDH1 R132H and HL60 IDH2 WT versus HL60 IDH2 R172K treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( B ) Synergy Map of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 R132H-5 (left) and in HL60 IDH1 R132H-11 (right) treated for 3 days with 0.25–4 µM of ATRA and 25–400 nM of VD alone or in combination. ( C ) Combination index of differentiation (CD11b expression induction) in HL60 IDH1 R132H-5 and in HL60 IDH1 R132H-11 treated for 3 days with ATRA and VD in combination with constant ratio of each drug. ( D ) Morphological characterization by MGG staining of HL60 IDH1 WT-2 versus HL60 IDH1 R132H-11 treated for 5 days with ATRA 1 µM and VD 100 nM alone or in combination. ( E ) HeatMap of CD11b expression and CD15 expression (MFI, ratio to untreated) measured by flow cytometry in 2HG-treated (100(U937)-200 µM for 1wk) HL60 IDH1 WT-2 , HL60 IDH1 WT-4 , HL60 IDH1 WT-7 , HL60, U937, THP1 and KG1a treated for 3 days with ATRA (0.1 µM for U937, 1 µM for others) or VD (100 nM) alone or in combination. ( F ) CD11b expression (MFI) measured by flow cytometry in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (left panel) and in HL60 IDH1 R132H (clone 5: ◇, clone 11: △) shCTL vs. shCEBPα (right panel) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( G ) CD11b expression (MFI) measured by flow cytometry in 2HG-treated HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (2HG 200 µM for 3 days) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. * p < 0.05; ** p < 0.01, *** p < 0.005.

Journal: Cancers

Article Title: Activation of Vitamin D Receptor Pathway Enhances Differentiating Capacity in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

doi: 10.3390/cancers13205243

Figure Lengend Snippet: Combine treatment with VD and ATRA produces a synergistic induction of differentiation in a CEBPα-dependent manner. ( A ) HeatMap of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 WT versus HL60 IDH1 R132H and HL60 IDH2 WT versus HL60 IDH2 R172K treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( B ) Synergy Map of CD11b expression (MFI, ratio to untreated) measured by flow cytometry in HL60 IDH1 R132H-5 (left) and in HL60 IDH1 R132H-11 (right) treated for 3 days with 0.25–4 µM of ATRA and 25–400 nM of VD alone or in combination. ( C ) Combination index of differentiation (CD11b expression induction) in HL60 IDH1 R132H-5 and in HL60 IDH1 R132H-11 treated for 3 days with ATRA and VD in combination with constant ratio of each drug. ( D ) Morphological characterization by MGG staining of HL60 IDH1 WT-2 versus HL60 IDH1 R132H-11 treated for 5 days with ATRA 1 µM and VD 100 nM alone or in combination. ( E ) HeatMap of CD11b expression and CD15 expression (MFI, ratio to untreated) measured by flow cytometry in 2HG-treated (100(U937)-200 µM for 1wk) HL60 IDH1 WT-2 , HL60 IDH1 WT-4 , HL60 IDH1 WT-7 , HL60, U937, THP1 and KG1a treated for 3 days with ATRA (0.1 µM for U937, 1 µM for others) or VD (100 nM) alone or in combination. ( F ) CD11b expression (MFI) measured by flow cytometry in HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (left panel) and in HL60 IDH1 R132H (clone 5: ◇, clone 11: △) shCTL vs. shCEBPα (right panel) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. ( G ) CD11b expression (MFI) measured by flow cytometry in 2HG-treated HL60 IDH1 WT (clone 2: ○, clone 7: ☐) shCTL vs. shCEBPα (2HG 200 µM for 3 days) treated for 3 days with ATRA (1 µM) and VD (100 nM) alone or in combination. * p < 0.05; ** p < 0.01, *** p < 0.005.

Article Snippet: For the generation of IDH2 WT or IDH2 R72K HL60, the following pSLIK vectors were used: pSLIK-IDH2-FLAG (Addgene plasmid #66806); pSLIK-IDH2-R172K-FLAG (Addgene plasmid #66807).

Techniques: Expressing, Flow Cytometry, Staining

Antibodies, lectins and proteins, as well as their molecular weights, transfer time and incubate time.

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Antibodies, lectins and proteins, as well as their molecular weights, transfer time and incubate time.

Article Snippet: Horseradish-peroxidase (HRP) conjugated Affinipure donkey anti-human IgG and rabbit anti-EEF1A2 were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Molecular Weight

Comparison of the binding ability of PVDF membrane and NC membrane to medium molecular weight proteins. ( a ) The pooled sera proteins (0.1–3.0 μg) were subjected to 8% SDS-PAGE. The electroblotted membranes are PVDF membrane (up) and NC membrane (down), respectively. The membranes were incubated with anti-alpha-1-acid glycoprotein (AGP), anti-eukaryotic transformation extension factor 1 alpha 2 (EEF1A2) and anti-transferrin (TF) antibodies. ( b ) Staining intensities were statistically analyzed (n = 3 individual experiments). Pink bar, PVDF membrane; Blue bar, NC membrane. Band intensities were analyzed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. N.S., not significant. All values are means ± S.E. (error bars).

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Comparison of the binding ability of PVDF membrane and NC membrane to medium molecular weight proteins. ( a ) The pooled sera proteins (0.1–3.0 μg) were subjected to 8% SDS-PAGE. The electroblotted membranes are PVDF membrane (up) and NC membrane (down), respectively. The membranes were incubated with anti-alpha-1-acid glycoprotein (AGP), anti-eukaryotic transformation extension factor 1 alpha 2 (EEF1A2) and anti-transferrin (TF) antibodies. ( b ) Staining intensities were statistically analyzed (n = 3 individual experiments). Pink bar, PVDF membrane; Blue bar, NC membrane. Band intensities were analyzed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. N.S., not significant. All values are means ± S.E. (error bars).

Article Snippet: Horseradish-peroxidase (HRP) conjugated Affinipure donkey anti-human IgG and rabbit anti-EEF1A2 were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Binding Assay, Molecular Weight, SDS Page, Incubation, Transformation Assay, Staining, Software

Sequences of primers

Journal: Bioscience Reports

Article Title: Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

doi: 10.1042/BSR20194191

Figure Lengend Snippet: Sequences of primers

Article Snippet: The Genetex and Proteintech anti-eEF1A2 antibodies detected a band in liver (data not shown) in spite of the absence of Eef1a2 at the mRNA level ( C,D).

Techniques:

Slope, intercept and correlation coefficient (R 2 ) output from SDS software to estimate efficiency of primers used for qPCR analyses

Journal: Bioscience Reports

Article Title: Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

doi: 10.1042/BSR20194191

Figure Lengend Snippet: Slope, intercept and correlation coefficient (R 2 ) output from SDS software to estimate efficiency of primers used for qPCR analyses

Article Snippet: The Genetex and Proteintech anti-eEF1A2 antibodies detected a band in liver (data not shown) in spite of the absence of Eef1a2 at the mRNA level ( C,D).

Techniques: Software

( A ) Schematic representation of the exon–intron organisation of eef1a1l1 (red), eef1a1a (black), eef1a1b (blue) and eef1a2 (yellow) structures obtained from the Ensembl database. Length (in base pairs) of exons and introns, which are not drawn to scale, are indicated above and below respectively. ( B ) Percentage identity matrix for zebrafish Eef1as at the nucleotide and amino acid sequence level (in brackets) calculated using Clustal Omega. ( C ) Phylogenetic relationship among the zebrafish Eef1as and other vertebrate eEF1As using the maximum likelihood method.

Journal: Bioscience Reports

Article Title: Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

doi: 10.1042/BSR20194191

Figure Lengend Snippet: ( A ) Schematic representation of the exon–intron organisation of eef1a1l1 (red), eef1a1a (black), eef1a1b (blue) and eef1a2 (yellow) structures obtained from the Ensembl database. Length (in base pairs) of exons and introns, which are not drawn to scale, are indicated above and below respectively. ( B ) Percentage identity matrix for zebrafish Eef1as at the nucleotide and amino acid sequence level (in brackets) calculated using Clustal Omega. ( C ) Phylogenetic relationship among the zebrafish Eef1as and other vertebrate eEF1As using the maximum likelihood method.

Article Snippet: The Genetex and Proteintech anti-eEF1A2 antibodies detected a band in liver (data not shown) in spite of the absence of Eef1a2 at the mRNA level ( C,D).

Techniques: Sequencing

The human eEF1A: heEF1A1 and heEF1A2, mouse eEF1A: meEF1A1 and meEF1A2. Identical and similar amino acid residues are indicated by black and grey backgrounds. Red asterisks (*) indicate some of the residues that are mutated in human eEF1A2, which are completely conserved in the four zebrafish eEF1A isoforms .

Journal: Bioscience Reports

Article Title: Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

doi: 10.1042/BSR20194191

Figure Lengend Snippet: The human eEF1A: heEF1A1 and heEF1A2, mouse eEF1A: meEF1A1 and meEF1A2. Identical and similar amino acid residues are indicated by black and grey backgrounds. Red asterisks (*) indicate some of the residues that are mutated in human eEF1A2, which are completely conserved in the four zebrafish eEF1A isoforms .

Article Snippet: The Genetex and Proteintech anti-eEF1A2 antibodies detected a band in liver (data not shown) in spite of the absence of Eef1a2 at the mRNA level ( C,D).

Techniques:

( A ) Expression of eef1a1l1 in different early embryonic stages detected by RT-PCR. The other eef1a genes were undetected at these stages (data not shown). ( B ) Expression of eef1a genes in 24, 48 and 72 hpf developmental stages using RT-PCR. NTC, no template control. ( C) Expression of eef1a genes in different tissues of adult zebrafish detected by RT-PCR. ( D ) Expression levels of eef1a genes in brain, muscle and liver tissues, and ( E ) comparison of the relative levels of their transcripts in these tissues. Expression values were normalised to those of ATPsynth, NADH and 16S . Results are means ± SD, n =3. * P <0.05; ** P <0.01; *** P <0.0001. For comparison, data were presented as the gene expression ratio of the target mRNA to the geometric mean of reference genes for each tissue. ( F ) Western blot showing Eef1a2 expression in brain, liver and muscle zebrafish tissues using eEF1A2-Abcam antibody (1:1000). Control is muscle tissue from mouse. ( G ) Validation of eEF1A2-Abcam antibody (ab82912) specificity using lysates isolated from individually transfected HEK293T cells with GFP-tagged Eef1a constructs. Abbreviation: Zf, zebrafish.

Journal: Bioscience Reports

Article Title: Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

doi: 10.1042/BSR20194191

Figure Lengend Snippet: ( A ) Expression of eef1a1l1 in different early embryonic stages detected by RT-PCR. The other eef1a genes were undetected at these stages (data not shown). ( B ) Expression of eef1a genes in 24, 48 and 72 hpf developmental stages using RT-PCR. NTC, no template control. ( C) Expression of eef1a genes in different tissues of adult zebrafish detected by RT-PCR. ( D ) Expression levels of eef1a genes in brain, muscle and liver tissues, and ( E ) comparison of the relative levels of their transcripts in these tissues. Expression values were normalised to those of ATPsynth, NADH and 16S . Results are means ± SD, n =3. * P <0.05; ** P <0.01; *** P <0.0001. For comparison, data were presented as the gene expression ratio of the target mRNA to the geometric mean of reference genes for each tissue. ( F ) Western blot showing Eef1a2 expression in brain, liver and muscle zebrafish tissues using eEF1A2-Abcam antibody (1:1000). Control is muscle tissue from mouse. ( G ) Validation of eEF1A2-Abcam antibody (ab82912) specificity using lysates isolated from individually transfected HEK293T cells with GFP-tagged Eef1a constructs. Abbreviation: Zf, zebrafish.

Article Snippet: The Genetex and Proteintech anti-eEF1A2 antibodies detected a band in liver (data not shown) in spite of the absence of Eef1a2 at the mRNA level ( C,D).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Transfection, Construct

( A ) Schematic showing outcross mating of founder (F0) fish and wild-type fish showing recovered F1 sequences (number of F1 fish for each allele are indicated in brackets, target sequences (yellow highlight) and PAM site (purple) with red showing inserted bases) and the predicted effect of Ins4 and Del2 mutant allele with aberrant residues shown in red (right). ( B ) No overt difference in homozygous Del2 (6 months) and homozygous Ins4 (8 months) adult fish from wild-type (6 months) adult fish. ( C ) The position of the three different primer sets; eef1a2S (blue triangle), eef1a2P (black triangle) and 3′eef1a2 (red triangle) is illustrated in relation to the gRNA target site (PAM site sequence in red). Expression levels of eef1a2 in homozygote Del2 (top panel) and homozygote Ins4 (bottom panel) fish using eef1a2P and 3′eef1a2 primer sets. Results were normalised to ATPsynth, NADH and 16S . (means ± S.E.M; n =3), * P <0.05. ( D ) Anti-GFAP antibody stained transverse sections of spinal cords of homozygous Del2 and Ins4 adult fish showed no sign of neurodegeneration. Negative control of a no primary (No Pri) was included which showed no staining. Scale bar = 100 µm. ( E ) Expression levels of eef1a1l1 (left), eef1a1a (middle) and eef1a1b (right) mRNA in homozygous Del2 (top panel) and Ins4 (bottom panel) adult brain. Data were normalised to ATPsynth, NADH and 16S and were presented as means ± S.E.M.; n =3.

Journal: Bioscience Reports

Article Title: Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish

doi: 10.1042/BSR20194191

Figure Lengend Snippet: ( A ) Schematic showing outcross mating of founder (F0) fish and wild-type fish showing recovered F1 sequences (number of F1 fish for each allele are indicated in brackets, target sequences (yellow highlight) and PAM site (purple) with red showing inserted bases) and the predicted effect of Ins4 and Del2 mutant allele with aberrant residues shown in red (right). ( B ) No overt difference in homozygous Del2 (6 months) and homozygous Ins4 (8 months) adult fish from wild-type (6 months) adult fish. ( C ) The position of the three different primer sets; eef1a2S (blue triangle), eef1a2P (black triangle) and 3′eef1a2 (red triangle) is illustrated in relation to the gRNA target site (PAM site sequence in red). Expression levels of eef1a2 in homozygote Del2 (top panel) and homozygote Ins4 (bottom panel) fish using eef1a2P and 3′eef1a2 primer sets. Results were normalised to ATPsynth, NADH and 16S . (means ± S.E.M; n =3), * P <0.05. ( D ) Anti-GFAP antibody stained transverse sections of spinal cords of homozygous Del2 and Ins4 adult fish showed no sign of neurodegeneration. Negative control of a no primary (No Pri) was included which showed no staining. Scale bar = 100 µm. ( E ) Expression levels of eef1a1l1 (left), eef1a1a (middle) and eef1a1b (right) mRNA in homozygous Del2 (top panel) and Ins4 (bottom panel) adult brain. Data were normalised to ATPsynth, NADH and 16S and were presented as means ± S.E.M.; n =3.

Article Snippet: The Genetex and Proteintech anti-eEF1A2 antibodies detected a band in liver (data not shown) in spite of the absence of Eef1a2 at the mRNA level ( C,D).

Techniques: Mutagenesis, Sequencing, Expressing, Staining, Negative Control