Journal: The Journal of Biological Chemistry

Article Title: The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB

doi: 10.1016/j.jbc.2022.101744

Figure Lengend Snippet: Generation of Ragulator-deficient clone with total mTORC1 activity. A , schematic representation of the findings of this study, depicting the phosphorylation of the TFEB substrate by mTORC1 bound to the Ragulator-Rag scaffold. B , the results of Western blotting studies on ΔRagulator and parental RAW264.7 cells. Lamtor1, Lamtor2, Lamtor3, Lamtor4, and Lamtor5 are components of the Ragulator complex; β-actin served as the internal control; mTOR and Raptor are components of mTORC1; RagC is necessary for the docking of TFEB to Ragulator. C , representative result of Western blotting studies for determining the activity of mTORC1. The control RAW264.7 cells and ΔRagulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. The phosphorylation of S6K in these cells was abolished following treatment with the mTOR kinase inhibitor, Torin1, at the indicated concentrations for 1 h. D , the densities of the p-S6K and S6K bands in panel C were quantified. The ratio of densities of the p-S6K band to S6K band is shown. Four independent experiments were performed. Bars indicate the median. Statistical significance was calculated using Mann–Whitney U test. E , the results of Upstream Regulator Analysis, as determined using the web-based software, Ingenuity Pathway Analysis. The pathways with z scores less than −2 are significantly inactivated in the ΔRagulator cells, compared to the RAW264.7 cells. The z score for the TFEB pathway was >2, which indicates that TFEB is significantly activated in the ΔRagulator cells than in the control RAW264.7 cells. F , the sizes of the ΔRagulator cells were larger than those of the parental RAW264.7 cells. The cell sizes of 1 × 10 4 cells were estimated using flow cytometry. Statistical significance was calculated using Welch’s t test. G , Western blotting for determining the phosphorylation of TSC2. Control RAW264.7 cells and ΔRagulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. Three biological triplicates were examined. H , the densities of the p-TSC2 and the TSC2 bands in panel G were quantified. The ratio of densities of the p-TSC2 band to TSC2 band is shown. Statistical significance was calculated using the Mann–Whitney U test. mTORC1, mammalian target of rapamycin complex 1; TFEB transcription factor EB.

Article Snippet: For Western blotting, the antibodies against S6K (#2708), p-S6K (T389, #9234), Akt (#4691), p-Akt (Ser473, #4060), 4E-BP1 (#9644), p-4E-BP1 (#2855), Lamtor1 (#8975), Lamtor2 (#8145), Lamtor3 (#8168), Lamtor4 (#12284), Lamtor5 (#14633), mTOR (#2983), p-mTOR (Ser2448, #5536), Raptor (#2280), RagC (#9480), Rheb (#13879), TSC2 (#4308), and p-TSC2 (Thr1462, #3617) were purchased from Cell Signaling Technology; while the antibodies against Lamin B (sc-6216), LAMP-1(clone 1D4B) and β-actin (clone AC-15) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Cell Culture, MANN-WHITNEY, Software, Flow Cytometry

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Recombinant, Sequencing, Modification, Membrane, Lysis, Transfection, Magnetic Beads, Software, Cytometry, In Vitro, cDNA Synthesis, Plasmid Preparation, Isolation, Mutagenesis, Silver Staining