sestrin1  (Proteintech)

Journal: Molecular cell

Article Title: Ring domains are essential for GATOR2-dependent mTORC1 activation

doi: 10.1016/j.molcel.2022.11.021

Figure Lengend Snippet: A. Flag-Sestrin2 was co-transfected with HA-WDR24, HA-WDR59, HA-Mios, HA-Seh1L, and HA-Sec13 in HEK293T cells. Cells were lysed and immunoprecipitated with HA beads, and the immunocomplexes were analyzed by immunoblotting as indicated. B. Wild-type, WDR24−/− and WDR59−/− cells were infected with HA-WDR59 constructs. The resulting cells were lysed and immunoprecipitated with HA beads, and the immunocomplexes were analyzed by immunoblotting with indicated antibodies. C. WDR24−/− cells were infected with HA-WDR24 wild-type and ΔRing constructs. Cells were lysed and immunoprecipitated with HA beads, and the immunocomplexes were analyzed by immunoblotting with indicated antibodies. D. The NPRL2HA knock-in HEK 293 cells were first infected with lentiviral constructs expressing Cas9-sgSESN1/2/3. Infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells. The resulting cells were deprived of leucine for 60 mins or starved and restimulated with leucine for 10 mins before harvesting for IP and IB analyses. E. In vitro poly-ubiquitination of NPRL2 by in vitro reconstituted GATOR2 (from recombinant proteins). F. Binding assay among different E2 enzymes with WDR24. 5 μg His6-tag E2 enzymes were immobilized on Ni-NTA beads and were incubated with 293T purified Flag-WDR24. The interaction was analyzed by immunoblotting with the Flag-antibody. G. Leucine restored the interaction of WDR24 with UBE2D3. HA-WDR24 and Flag-Sestrin2 or Flag-Sestrin2 L261A mutant were co-transfected into Sestrin1–3 triple-null HEK 293T cells. Cells were deprived of full amino acids for 60 min, and leucine (100 μM) was added directly to the immunoprecipitants. After re-washing, the immunocomplex was incubated with UBE2D3, and the interaction was analyzed by immunoblotting with the indicated antibody. H. Sestrin1–3 triple-null cells expressing Sestrin2 wild-type, L261A (do not bind to leucine), S190W (do not bind to WDR24) were infected with HA-NPRL2 expressing virus and selected with puromycin (1 μg/mL). Cells were then transfected with Myc-Ub, and resulting cells were deprived of leucine for 60 min or starved and restimulated with leucine for 10 min. The ubiquitination of NPRL2 was analyzed as in (D). I. WDR24−/− in NPRL2HA background were reconstituted with indicated constructs. The mTORC1 activation and ubiquitination level of NPRL2 was analyzed. J. A schematic depicting Sestin2 and Mios/WDR24 Ring interaction mode in response to leucine availability. Upon leucine stimulation, Sestrin2 dissociates from the Ring domain of WDR24 accompanied by WDR24-Mios Ring conformational changes, leading to a Ring-Ring semi-open status, WDR24 ubiquitinates NPRL2. In the status of leucine starvation, Sestin2 and Mios bind to the Ring domain of WDR24, leading to a closed Ring-Ring interaction mode, in which the E3 activity of WDR24 is blocked. Other E3 ligase might interacts with GATOR2 to dictate amino-acids mediated NPRL2 ubiquitiantion. See also Figure S5.

Article Snippet: Rabbit anti-Sestrin1 , Proteintech , Cat#: 21668.

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Construct, Knock-In, Expressing, In Vitro, Recombinant, Binding Assay, Incubation, Purification, Mutagenesis, Virus, Activation Assay, Activity Assay

Journal: Molecular cell

Article Title: Ring domains are essential for GATOR2-dependent mTORC1 activation

doi: 10.1016/j.molcel.2022.11.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Sestrin1 , Proteintech , Cat#: 21668.

Techniques: Virus, Recombinant, Triple Knockout, Knock-In

Journal: Journal of Ovarian Research

Article Title: Sestrin1, 2, and 3 are dispensable for female fertility in mice

doi: 10.1186/s13048-024-01345-z

Figure Lengend Snippet: Construction of Sesn1 −/− , Sesn2 −/− , and Sesn3 −/− mice. ( A ) Limiting the expression of gene fragments for Sestrin1 , Sestrin2 , and Sestrin3 using the CRISPR/Cas9 system. ( B ) Genotyping of wild-type (WT) and knockout mice. ( C ) Detection of S esn1, Sesn2 and Sesn3 mRNA in Sesn1 −/− , Sesn2 −/− , and Sesn3 −/− and WT mice ovaries by Agarose gel electrophoresis of real time PCR. ( D ) Expression of SESN1, 2, and 3 in WT and knockout mice ovaries detected by IHC. Scale bar = 50 μm

Article Snippet: Ovarian tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval in 10 mM citrate buffer at 95 °C for 20 min. Primary antibodies, including Anti-Sestrin1 (Novus, NBP2-20316), Anti-Sestrin2 (Proteintech,10759-1-AP), and Anti-Sestrin3 (Proteintech,11431-2-AP), were applied at a 1:100 dilution and incubated overnight at 4 °C following the inhibition of endogenous peroxidase activity.

Techniques: Expressing, CRISPR, Knock-Out, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction