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

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

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

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

Journal: bioRxiv

Article Title: A Highly Efficient and Faithful MDS Patient-Derived Xenotransplantation Model for Pre-Clinical Studies

doi: 10.1101/265082

Figure Lengend Snippet: MISTRG replicate granulocytic and megakaryocytic differentiation in response to inhibition of mutant IDH2. In vivo treatment of mutant IDH2 R140Q in MDS-EB-2 (Y021) engrafted MISTRG mice with the IDH2 MUT inhibitor enasidenib. ( a ) Representative histologic images of vehicle (n=8, left) and enasidenib (n=6, right) treated mice engrafted with MDS-EB-2 (Y021). IHC stains for huCD45, huCD68, huCD15, and huCD61 (scale bars 10μm, original magnification 60X). ( b ) Representative FACS plots showing myeloid maturation in response to enasidenib and quantitation of huCD15+ and huCD11b+ expression in vehicle versus enasidenib treated MISTRG mice. ( c ) Comparison of human engraftment in BM from vehicle (n=8) and enasidenib (n=6) treated MISTRG mice. ( d ) Quantitation of huCD41+ expression in PB and BM from vehicle (n=8) and enasidenib (n=6) treated MISTRG mice ( e ) Quantitation of D-2HG in plasma of pre- and post-administration of Vehicle or Enasidenib. Individual mice are represented by symbols with mean ± S.E.M.; statistics represent Mann Whitney test; n.s. not significant, *p<0.05, **p<0.01 for aggregate NSG vs. MISTRG. ( f ) Representation of VAFs of driver mutations in vehicle (left) or enasidenib (right) treated MISTRG (y-axis) plotted against the patient’s VAFs (x-axis). Individual mice represented by symbol shape, mutations color coded. Linear regressors, pearson correlations and p-values between patient and xenograft VAF are displayed.

Article Snippet: To generate isogenic wildtype and mutant expressing cell lines the lentiviral plasmids pSLIK-IDH2-FLAG and pSLIK-IDH2-R172K-FLAG (a gift from Christian Metallo, Addgene plasmid ## 66806, 6680, ) were used to transduce human erythroid leukemia cell (HEL) cell lines.

Techniques: Inhibition, Mutagenesis, In Vivo, Quantitation Assay, Expressing, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression

doi: 10.1038/bjc.2011.500

Figure Lengend Snippet: Quantification of total eEF1As and of eEF1A1/2 proteins in cytoplasmic and cytoskeletal/nuclear-enriched fractions. ( A , B ) Quantification of total eEF1As and eEF1A1/2 proteins in cytoplasmic extract. The blots were treated by either eEF1A ( A ) or eEF1A1/eEF1A2 antibodies ( B ). The quantifications are expressed as mean values±s.e.m., n =6 for eEF1A and n =3–10 for eEF1A1 and eEF1A2. The asterisks mark statistical significance: * eEF1A and # eEF1A2 with respect to PZHPV-7. Representative blots are shown below the graphs. ( C and D ) Quantification of total eEF1As and eEF1A1/2 proteins in cytoskeletal/nuclear extract. The blots were treated by either eEF1A ( C ) or eEF1A1/eEF1A2 antibodies ( D ). The quantifications are expressed as mean values±s.e.m., n =4 for eEF1A and n =3–8 for eEF1A1 and eEF1A2. The significance is indicated by * eEF1A, § eEF1A1 and # eEF1A2 with respect to PZHPV-7. Representative blots are shown below the graphs.

Article Snippet: Prostate cell lines were plated on Thermanox plastic cover-slips (Nunc, Rochester, NY, USA), fixed with 1% paraformaldehyde in HBBS buffer (Hepes 10 mM, NaCl 150 mM, KCl 5 mM, CaCl 2 1.8 mM) for 30 min, RT and permeabilised with ice-cold methanol at 4°C, 2 min. After blocking in 0.5% bovine serum albumin, samples were incubated overnight at 4°C with anti-rabbit eEF1A2 polyclonal antibody (ProteinTech Group Inc., Chicago, IL, USA) rinsed with HBBS and incubated with an anti-rabbit IgG antibody conjugated to FITC (DAKO A/S, Glostrup, Denmark) for 1 h, RT.

Techniques:

Journal: British Journal of Cancer

Article Title: Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression

doi: 10.1038/bjc.2011.500

Figure Lengend Snippet: eEF1A2 IF. The exponentially growing cell lines indicated were plated on plastic cover-slips and fixed before the overnight incubation with eEF1A2 polyclonal antibody whose was revealed by an anti-rabbit IgG conjugated with FITC to fluorescence examination as described in Materials and Methods. ( A , C , E and G ) Contrast microscopy (the bar marks 50 μ M ); ( B , D , F and H ) fluorescence microscopy.

Article Snippet: Prostate cell lines were plated on Thermanox plastic cover-slips (Nunc, Rochester, NY, USA), fixed with 1% paraformaldehyde in HBBS buffer (Hepes 10 mM, NaCl 150 mM, KCl 5 mM, CaCl 2 1.8 mM) for 30 min, RT and permeabilised with ice-cold methanol at 4°C, 2 min. After blocking in 0.5% bovine serum albumin, samples were incubated overnight at 4°C with anti-rabbit eEF1A2 polyclonal antibody (ProteinTech Group Inc., Chicago, IL, USA) rinsed with HBBS and incubated with an anti-rabbit IgG antibody conjugated to FITC (DAKO A/S, Glostrup, Denmark) for 1 h, RT.

Techniques: Incubation, Fluorescence, Microscopy

Journal: British Journal of Cancer

Article Title: Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression

doi: 10.1038/bjc.2011.500

Figure Lengend Snippet: RT–PCR of EEF1A1/2 in human biopsy samples. ( A ) Dissection of Finefix-fixed paraffin-embedded samples. Prostate tissues histologic haematoxylin–eosin-stained sections of tissues from patient 1 are illustrated as an example ( × 20 magnification): normal tissue, hyperplastic peritumoural tissue, neoplastic tissue. ( B ) RT–PCR of Finefix-fixed paraffin-embedded samples. (1) Normal tissue, (2) hyperplatic peritumoural tissue, (3) neoplastic tissue from patient 1, (4) hyperplastic peritumoural tissue, (5) neoplastic tissue from patient 2, (6) hyperplastic peritumoural tissue, (7) neoplastic tissue from patient 3, (8) hyperplastic peritumoural tissue, (9) neoplastic tissue from patient 4 and (10) LoVoDX-positive control. ( C ) RT–PCR of human benign adenoma. The cDNA of fresh benign adenoma was amplified by using EEF1A1 primer pair giving amplicon of 229 bp (lanes 1 and 2) or EEF1A2 primer pair giving amplicon of 183 bp (lanes 3 and 4); lanes 2 and 4, adenoma; lanes 1 and 3, HepG2-positive control. ( D ) EEF1A2 RT–PCR on tissue archive formalin-fixed paraffin-embedded tissues. The samples were amplified with primer pair giving an amplicon of 91 bp. Lanes 1, 2, 4, 5, 7, 8, 9, 10 and 12 cancer samples; lanes 3, 6, 11 and 13 perineoplastic tissues; lane C+ positive control HepG2 cells; C− benign hyperplasia. The arrows mark the specific amplicon. ( E ) Probing of the amplicons on archive formalin-fixed paraffin-embedded tissues. The RT–PCR products shown in ( B ) were used to perform dot blotting with the specific EEF1A2 or 28S rRNA probes. Lanes 1, 2, 4, 5, 7, 8, 9, 10 and 12 prostate cancer samples; lanes 3, 6, 11 and 13, perineoplastic tissues; C− benign hyperplasia; C+ positive control HepG2, Co control of RT–PCR paraffin block.

Article Snippet: Prostate cell lines were plated on Thermanox plastic cover-slips (Nunc, Rochester, NY, USA), fixed with 1% paraformaldehyde in HBBS buffer (Hepes 10 mM, NaCl 150 mM, KCl 5 mM, CaCl 2 1.8 mM) for 30 min, RT and permeabilised with ice-cold methanol at 4°C, 2 min. After blocking in 0.5% bovine serum albumin, samples were incubated overnight at 4°C with anti-rabbit eEF1A2 polyclonal antibody (ProteinTech Group Inc., Chicago, IL, USA) rinsed with HBBS and incubated with an anti-rabbit IgG antibody conjugated to FITC (DAKO A/S, Glostrup, Denmark) for 1 h, RT.

Techniques: Reverse Transcription Polymerase Chain Reaction, Dissection, Staining, Positive Control, Amplification, Formalin-fixed Paraffin-Embedded, Blocking Assay