Fig 1: The expression of YBX1, EEF1A2, PCLO and ZYX in the eluent of the biotin-labeled ZYX36-58 and scrambled peptide pull down lysate. Input: the total protein of biotin labeled ZYX36-58 and scrambled peptide treated SKOV3 cells, Output: The protein which could not interact with the biotin labeled ZYX36-58 and scrambled peptide, pull down: the protein which interacts with the biotin labeled ZYX36-58 and scrambled peptide.
Fig 2: eEF1A2 interacts with circCTDP1 to regulate PFKFB3.a VSMCs were transfected with the indicated constructs. PFKFB3, flag and ß-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 ß-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.
Fig 3: KLF4 upregulates PFKFB3 expression via eEF1A2.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 ß-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 ß-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 ß-actin (n = 3 independent experiments, error bars show SEM). f VSMCs were transfected with the indicated constructs. PFKFB3, eEF1A2 and ß-actin protein levels were measured by immunoblotting and PFKFB3 protein levels were quantified by normalizing to ß-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.
Fig 4: Loss of NOP2/Sun RNA methyltransferase family members 6 inhibits the proliferation of osteosarcoma in vivo. (A) Representative image of all the nude mice from different groups. The tumors were on the nude mice. (B) Representative images of all the tumors collected from nude mice from different groups. (C) Weight and (D) Volume of all the tumors. (E) Result of immunohistochemistry (scale bar, 50 µM). ***P#x003C;0.001. EEF1A2, eukaryotic elongation factor 1 a-2; sh, short-hairpin; ctrl, control.
Fig 5: EEF1A2 is the potential target of NSUN6 in human OS. (A) Volcano plot showing that expression difference of EEF1A2, PRAME, FGFBP2 and TP53 was the largest between high- and low-NSUN6 groups. Correlation of NSUN6 with (B) EEF1A2 (P=0.0017), (C) PRAME (P=0.028), (D) FGFBP2 (P=0.028) and (E) TP53 (P=0.086) in human OS. Expression of EEF1A2 mRNA in sh-ctrl, sh-NSUN6#1 group and sh-NSUN6#2 groups of (F) 143b and (G) U2-OS cells. RIP assay results in (H) 143b and (I) U2-OS cells. IgG was used as a negative control. ***P#x003C;0.001. NSUN6, NOP2/Sun RNA methyltransferase family member 6; OS, osteosarcoma; EEF1A2, eukaryotic elongation factor 1 a-2; PRAME, melanoma antigen preferentially expressed in tumors; FGFBP2, fibroblast growth factor binding protein 2; sh, short-hairpin; ctrl, control.
Supplier Page from Proteintech Group Inc for EEF1A2 antibody