methyl β d glucuronide  (Biosynth Carbosynth)

Journal: The Journal of biological chemistry

Article Title: Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity.

doi: 10.1016/j.jbc.2022.102633

Figure Lengend Snippet: Figure 4. The induction of HIF1α target genes expression and stabilization of HIF1α by SMYD3 are independent of HIF1α hydroxylation and pVHL intactness. A–C, qPCR analysis of GLUT1 (A), PGK1 (B), and PDK1 (C) mRNA in HEK293T cells transfected with or without pCMV-SMYD3 for 24 h, followed by treatment with DMSO or FG4592 (100 μM) for 8 h. EV, pCMV empty vector (control). Data show mean ± SD; Student’s two-tailed t test. *p < 0.05, **p < 0.01. Data from three independent experiments. D–F, qPCR analysis of GLUT1 (D), PDK1 (E), and VEGF (F) mRNA in VHL-deficient HEK293T cells (VHL−/−) transfected with an increasing amount of pCMV-SMYD3 expression plasmid. pCMV empty vector was used as a control (-). Data show mean ± SD; Student’s two-tailed t test. **p < 0.01, ***p < 0.001, ****p < 0.0001. Data from three independent experiments. G, immunoblotting of endogenous Hif1α expression in Smyd3- deficient or wildtype MEF cells (Smyd3−/−or Smyd3+/+) treated with an increasing amount of FG4592 for 6 h. H, the relative intensities of Hif1α in (G) determined by normalizing the intensities of Hif1α to the intensities of Gapdh. I, immunoblotting of endogenous Hif1α expression in Smyd3-deficient or wildtype MEF cells (Smyd3−/−or Smyd3+/+) treated with an increasing time of FG4592 (100 μM) for 0 to 6 h. J, the relative intensities of Hif1α in (I) determined by normalizing the intensities of Hif1α to the intensities of Gapdh. HIF, hypoxia-inducible factor; MEF, mouse embryonic fibroblast; qPCR, quantitative RT–PCR; VHL, von Hippel-Lindau.

Article Snippet: Anti-Myc (#SC-40) and anti-GAPDH (#SC-477242) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test, Western Blot, Quantitative RT-PCR

Journal: The Journal of biological chemistry

Article Title: Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity.

doi: 10.1016/j.jbc.2022.102633

Figure Lengend Snippet: Figure 5. SMYD3 stabilizes and activates HIF1α independent of its methyltransferase activity. A and B, qPCR analysis of PGK1 (A) and PDK1 (B) mRNA in HEK293T cells transfected with expression plasmids encoding wildtype SMYD3 or its enzymatically dead mutant SMYD3-F183A (HA empty vector [EV] was used as a control) under normoxia (21% O2) or hypoxia (1% O2) for 24 h. Data show mean ± SD; Student’s two-tailed t test. ns, not significant, *p < 0.05, **p < 0.01. Data from three independent experiments. C, co-immunoprecipitation of HA-SMYD3-F183A with Myc-HIF1α. HEK293T cells were cotransfected with indicated plasmids for 24 h. Anti-HA antibody-conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. D and E, immunoblotting of exogenous Myc-HIF1α expression in HEK293T (D) or H1299 (E) cells transfected with expression plasmids encoding wildtype SMYD3 or its enzymatically dead mutant SMYD3-F183A (HA empty vector [-] was used as a control). The relative intensities of HIF1α were determined by normalizing the intensities of HIF1α to the intensities of GAPDH. Data show mean ± SD; Student’s two- tailed t test. ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data from three independent experiments. qPCR, quantitative RT–PCR; HIF, hypoxia- inducible factor.

Article Snippet: Anti-Myc (#SC-40) and anti-GAPDH (#SC-477242) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Control, Two Tailed Test, Immunoprecipitation, Western Blot, Quantitative RT-PCR