antibodies flag epitope (m2  (Millipore)

Journal: Nature Communications

Article Title: An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway

doi: 10.1038/s41467-024-46680-3

Figure Lengend Snippet: a Mass spectrometric analyses identify Unmet-derived peptides in immunoprecipitates from S2R+ cells expressing FLAG-tagged Mio, a component of the dGATOR2 complex. Unmet and previously known components of the mTORC1 pathway are colored by normalized peptide representation according to the scale below. b Recombinant Unmet co-immunoprecipitates endogenous GATOR1 and GATOR2 components in S2R+ cells. Anti-HA immunoprecipitates were prepared from S2R+ cells bearing endogenous FLAG knock-in tags at either the Iml1 (dGATOR1) or the dWDR59 (dGATOR2) locus, and transfected with the indicated cDNAs in copper-inducible metallothionein (MT) expression vectors. Following a 48-h induction with 75 μM CuSO 4 , cell lysates and immunoprecipitates were analyzed by immunoblotting for levels of the relevant epitope tags. HA-Und served as a negative control. c Recombinant Unmet interacts with dGATOR2, but not dGATOR1 or the corresponding human complexes. Anti-HA immunoprecipitates were collected from HEK-293T cells co-transfected with the indicated cDNAs in expression vectors and analyzed alongside cell lysates as in ( b ). d Deprivation of methionine, but not leucine, enhances the interaction between Unmet and dGATOR2. HEK-293T cells transiently expressing FLAG-tagged dGATOR2 and the indicated HA-tagged cDNAs were cultured in full RPMI or RPMI lacking leucine or methionine for 1 h. FLAG immunoprecipitates and cell lysates were analyzed by immunoblotting for the levels of the relevant proteins. e SAM, but not amino acids, disrupts the interaction between Unmet and dGATOR2 in vitro. FLAG immunoprecipitates were prepared from HEK-293T cells transfected with the indicated cDNAs. A mixture containing 1 mM of each amino acid or 1 mM of SAM was added directly to the immunoprecipitates. FLAG immunoprecipitates and cell lysates were analyzed as in ( d ). f Unmet binds SAM with a K d of 9.6 μM. Purified FLAG-Unmet protein was analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining. Binding assays contained 10 μg of purified FLAG-Unmet, 5 μM [ 3 H]SAM, and the indicated concentrations of unlabeled SAM. Values for each point represent the means ± s.d. of three technical replicates from one representative experiment. Binding experiments were repeated three times.

Article Snippet: Reagents were obtained from the following sources: antibody against the FLAG M2 epitope (F1804) from Millipore Sigma; antibody against Raptor (09-217) from EMD Millipore; HRP-labeled anti-mouse IgG (7076) and anti-rabbit IgG (7074) secondary antibodies from Cell Signaling Technology; antibodies against β-actin (4967), phospho-T398 dS6K (9209), Mios (13557), cleaved Drosophila Dcp-1 Asp216 (9578), FLAG epitope tag (14793), HA epitope tag (3724), and myc epitope tag (2278) from Cell Signaling Technology; antibody against hu-li tai shao (1B1) from the Developmental Studies Hybridoma Bank (DSHB); antibody against Depdc5 (ab185565) from Abcam; Alexa 488 and 555-conjugated secondary antibodies from Thermo Fisher Scientific.

Techniques: Derivative Assay, Expressing, Recombinant, Knock-In, Transfection, Western Blot, Negative Control, Cell Culture, In Vitro, Purification, Polyacrylamide Gel Electrophoresis, Staining, Binding Assay

Journal: Nature Communications

Article Title: An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway

doi: 10.1038/s41467-024-46680-3

Figure Lengend Snippet: a Recombinant CARNMT1, the human homolog of Unmet, interacts with dGATOR2 but not its human counterpart. Anti-HA immunoprecipitates from HEK-293T cells expressing the indicated cDNAs were analyzed as in Fig. . FLAG-metap2 served as a negative control. b Recombinant S. pombe CARNMT1, the fission yeast homolog of Unmet, interacts with dGATOR2 but not the S. pombe GATOR2 complex (SEACAT). Anti-FLAG immunoprecipitates from HEK-293T cells expressing the indicated cDNAs were analyzed as in Fig. . c Schematic of the interactions between homologs of Unmet and GATOR2 in three species. d Rapid evolution of the Mio sequence in Dipterans corresponds to the acquisition of Unmet binding. A maximum likelihood phylogenetic tree constructed using Mio protein sequences from 12 species was matched to the results of binding assays between Unmet and GATOR2 homologs, as assayed in Supplementary Fig. . Mio diverged so sharply in Dipterans that arthropod sequences from outside the order cluster with vertebrate sequences, in contrast to the topology of a classical species tree, shown in Supplementary Fig. . Node labels indicate bootstrap support values. Scale bar, 0.1 substitutions per site. e Dipteran-specific residues on Mio (magenta) are surface-exposed and map to flexible loops on the beta-propeller of Mio. Green cartoon, human Mios; orange cartoon, human Seh1L; derived from the structure of the full human GATOR2 complex (PDB: 7UHY). Dipteran-specific residues are annotated on the alignment in Supplementary Fig. .

Article Snippet: Reagents were obtained from the following sources: antibody against the FLAG M2 epitope (F1804) from Millipore Sigma; antibody against Raptor (09-217) from EMD Millipore; HRP-labeled anti-mouse IgG (7076) and anti-rabbit IgG (7074) secondary antibodies from Cell Signaling Technology; antibodies against β-actin (4967), phospho-T398 dS6K (9209), Mios (13557), cleaved Drosophila Dcp-1 Asp216 (9578), FLAG epitope tag (14793), HA epitope tag (3724), and myc epitope tag (2278) from Cell Signaling Technology; antibody against hu-li tai shao (1B1) from the Developmental Studies Hybridoma Bank (DSHB); antibody against Depdc5 (ab185565) from Abcam; Alexa 488 and 555-conjugated secondary antibodies from Thermo Fisher Scientific.

Techniques: Recombinant, Expressing, Negative Control, Sequencing, Binding Assay, Construct, Derivative Assay

Journal: Nature Communications

Article Title: An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway

doi: 10.1038/s41467-024-46680-3

Figure Lengend Snippet: a Human CARNMT1 can act as a negative regulator of mTORC1 signaling when human GATOR2 is replaced with the fly GATOR2 complex. Mios-deficient HEK-293T cells expressing the indicated cDNAs were starved in RPMI lacking amino acids for 1 h and then restimulated with amino acids for 15 min. Anti-FLAG immunoprecipitates were analyzed as in Fig. . b Evolutionary model for co-option of ligand-binding proteins by GATOR2. c Phylogenetic tree representing the evolution of nutrient sensing capabilities in the mTORC1 pathway. Conserved core components of the mTORC1 pathway are shown as white circles, connected by lines that represent protein-protein interactions. Orthologs share the same color. Dark gray blobs highlight species-restricted interactions between nutrient sensors and core components of the mTORC1 pathway. The eukaryotic nutrient sensors may ultimately share evolutionary origins with prokaryotic enzymes (shown as diamonds).

Article Snippet: Reagents were obtained from the following sources: antibody against the FLAG M2 epitope (F1804) from Millipore Sigma; antibody against Raptor (09-217) from EMD Millipore; HRP-labeled anti-mouse IgG (7076) and anti-rabbit IgG (7074) secondary antibodies from Cell Signaling Technology; antibodies against β-actin (4967), phospho-T398 dS6K (9209), Mios (13557), cleaved Drosophila Dcp-1 Asp216 (9578), FLAG epitope tag (14793), HA epitope tag (3724), and myc epitope tag (2278) from Cell Signaling Technology; antibody against hu-li tai shao (1B1) from the Developmental Studies Hybridoma Bank (DSHB); antibody against Depdc5 (ab185565) from Abcam; Alexa 488 and 555-conjugated secondary antibodies from Thermo Fisher Scientific.

Techniques: Expressing, Ligand Binding Assay, Protein-Protein interactions