gtp rheb antibody  (Proteintech)

Journal: Scientific Reports

Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy

doi: 10.1038/s41598-018-38473-8

Figure Lengend Snippet: RV infection downregulates let-7g expression. ( A ) HT29 cells were mock treated or infected with RV for indicated time points. Whole cell lysates were prepared, followed by western blot analysis using specific antibodies against Rheb-GTP, TSC1, TSC2, and RV-VP6. The blot was reprobed with an antibody to GAPDH for normalization. ( B ) In silico analysis of the TSC1 3′UTR revealed a single putative let-7g binding site. The let-7g target region of TSC1 (GenBank accession number NM_000368) is indicated. ( C ) HT29 cells were transfected with mimic let-7g (40 nM) (left panel) or pmR-ZsGreen1-pre-let7g (right panel) followed by western blot analysis using specific antibody against TSC1. Relative fold differences of the TSC1 level was analyzed after normalization with GAPDH from at least three independent experiments. ( D ) HT29 cells were mock treated or infected with RV-SA11 for indicated time points. Total RNA from mock- or virus-infected cells was extracted and expression of let-7g was analyzed by qRT-PCR and normalized to the expression of U6 snRNA. ( E ) RV-VP6 RNA level was measured and plotted as relative RNA level in comparison to mock-infected cells. ( F ) Cells were infected with UV inactivated RV-SA11. At 8hpi the total RNA was extracted and the relative expression of let-7g was quantified by qRT-PCR analysis. ( G ) VP6 RNA level was measured as a marker of viral infection in UV-inactivated RV-SA11 infected cells. Results are presented as the means and standard deviations from at least two independent experiments.

Article Snippet: The membranes were probed with antibodies to mTOR (#2972), p-mTOR (#2971), TSC1 (#4906), Rheb-GTP (#13879), LC3I/II (#12741), Beclin1 (#3495), ATG5 (#12994) (Cell Signaling), TSC2 (#sc-893) (Santa Cruz, USA) RV-VP6 (HyTest: 3C10) or RV-NSP3 (kind gift from Prof. Koki Taniguchi).

Techniques: Infection, Expressing, Western Blot, In Silico, Binding Assay, Transfection, Virus, Quantitative RT-PCR, Comparison, Marker

Journal: Scientific Reports

Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy

doi: 10.1038/s41598-018-38473-8

Figure Lengend Snippet: Schematic diagram showing the crosstalk between miR-99b, let-7g, and RV. RV infection downregulates let-7g which in turn elevates TSC1 expression. Inhibition of let-7g also promotes TSC1 mediated suppression of Rheb-GTP expression. On the other hand, RV infection upregulates miR-99b that directly targets mTOR. RV mediated upregulation of miR-99b and suppression of Rheb-GTP leads towards containment of mTOR expression. These results lead towards activation of autophagy mediators during RV infection. Arrows denote upregulation, and blunt arrows denote inhibition.

Article Snippet: The membranes were probed with antibodies to mTOR (#2972), p-mTOR (#2971), TSC1 (#4906), Rheb-GTP (#13879), LC3I/II (#12741), Beclin1 (#3495), ATG5 (#12994) (Cell Signaling), TSC2 (#sc-893) (Santa Cruz, USA) RV-VP6 (HyTest: 3C10) or RV-NSP3 (kind gift from Prof. Koki Taniguchi).

Techniques: Infection, Expressing, Inhibition, Activation Assay