Journal: Molecular cell

Article Title: Translational control through differential ribosome pausing during amino acid limitation in mammalian cells

doi: 10.1016/j.molcel.2018.06.041

Figure Lengend Snippet: KEY RESOURCES TABLE :

Article Snippet: pADHS2: AAVS1-CAG-RagBWT , Cloned from Qian et al., 2014 and Sancak et al. 2008 , Cloned Flag-RagB-WT from Flag pLJM1 RagB wt, Addgene #19313, into AAVS1-CAG-hrGFP.

Techniques: Recombinant, Staining, Western Blot, Stripping, Hybridization, SYBR Green Assay, Northern Blot, Homologous Recombination, Clone Assay, Sequencing, FLAG-tag, Software

Journal: bioRxiv

Article Title: p27 regulates the autophagy-lysosomal pathway via the control of Ragulator and mTOR activity in amino acid deprived cells

doi: 10.1101/2020.01.07.896860

Figure Lengend Snippet: (A) LAMTOR1 was immunoprecipitated with anti-HA antibodies from HEK293 cell expressing HA-LAMTOR1 and/or Flag-RagB and/or p27 and immunoblotted against Flag (RagB) and HA (LAMTOR1). Expression of the transfected proteins was verified by immunoblot of extracts with anti-Flag, −p27 and −HA antibodies. (B) Quantification of the amount of RagB co-precipitated with LAMTOR1 as in A from 5 experiments. Values were normalized to that in absence of p27. (C) RagB/D were immunoprecipitated with anti-Flag antibodies from HEK293 cell expressing HA-LAMTOR1 and/or Flag-RagB/D and/or Flag-RagB Q99L (RagB GTP)/RagD S77L (RagD GDP) and/or p27 and immunoblotted against Flag (RagB/D) and HA (LAMTOR1). Expression of transfected p27 and HA-LAMTOR1 in corresponding extracts are shown. (D) RagC was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR4 co-precipitated was determined. Control IP with rabbit IgG was used as control. Levels of RagC and LAMTOR4 in the corresponding extracts are shown. β-actin was used as loading control. (E) Ratio of LAMTOR4 co-precipitated by that of RagC immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h, normalized to full medium condition from 2 experiments as described in E. (F) RagB was immunoprecipitated with anti-Flag antibodies from HEK293 cells expressing Myc-Raptor and/or Flag-RagB and/or p27 and immunoblotted against Myc (Raptor) and Flag (RagB). Expression of the transfected proteins was verified by immunoblot of extracts with anti-Myc, −p27 and −Flag antibodies. (G) Quantification of the amount of Raptor co-precipitated with RagB as described in G from 3 experiments. Values were normalized to that in absence of p27. (H) Raptor was immunoprecipitated with anti-Myc antibodies from HEK293 cells expressing Myc-Raptor and/or Flag-RagB Q99L (RagB GTP)/RagD S77L (RagD GDP) and/or p27 and immunoblotted against Flag (RagB/D) and Myc (Raptor). Expression of transfected p27, Flag RagB/D and Myc-Raptor in corresponding extracts are shown. (I) Schematic summarizing the impact of p27 on Rag and mTORC1 recruitment to Ragulator. (J) TFEB immunostaining in p27 +/+ and p27 −/− MEFs in full medium (0 h) or aa-starved for 48 h. F-actin was stained with phalloïdin and DNA with Hoechst. Scale bars are 50 µm. (K) Percentage of cells with nuclear TFEB signal in cells treated as in K from 3 experiments. At least 87 cells per condition for each genotype were analyzed in each experiment. (L) Fold change of the v-ATPase subunit ATP6V0E1, CTSB (Cathepsin-B) and PUMA mRNA levels in p27 +/+ and p27 −/− MEFs in full medium or aa starved for 1 h or 48 h, determined by RT-qPCR and normalized to GAPDH levels from 8 experiments. All values were normalized to p27 +/+ MEFs in full medium (0 h). (M) Immunoblot for the v-ATPase subunit ATP6V1B1/2, PUMA and p27 in p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h. Grb2 was used as loading control. (B, E, G, K, L) Bar graphs show means ± SEM. Statistical significance was evaluated by unpaired t-test with Welch’s correction (B, G) or 2-way ANOVA (K, L); ns: p >0.05; *: p ≤ 0.05; ***: p ≤ 0.001; ****: p ≤ 0.0001.

Article Snippet: Recombinant His-tagged LAMTOR1 (#CSB-EP757083HU) was purchased from Cusabio Technology LLC. p27 constructs and p27 point mutants and deletion mutants in pCS2+, pcDNA3.1+Hygro (Invitrogen), pQCXIP (Clontech), pBabe-puro, pWZL-Blast and pGEX4T1 were described previously , , . pBabe-puro-mCherry-eGFP-LC3B was a gift from Jayanta Debnath (Addgene #22418) . pRK5 Flag-p18 (LAMTOR1) (Addgene #42331), pRK5 HA-p18 (LAMTOR1) (Addgene #42338), pRK5 HA C7orf59 (LAMTOR4) (Addgene #42336), pRK5 Flag C7orf59 (LAMTOR4) (Addgene #42332), pRK5 Flag p14 (LAMTOR2) (Addgene #42330), pRK5 HA mp1 (LAMTOR3) (Addgene #42329), pRK5 HA HBXIP (LAMTOR5) (Addgene #42328) and pRK5 Flag HBXIP (LAMTOR5) (Addgene #42326) , pRK5 Myc-Raptor (Addgene #1859) , and pLJM1 Flag-RagB (Addgene #19313), pLJM1 Flag-RagB Q99L (Addgene #19315), pLJM1 Flag-RagD (Addgene #19316), pLJM1 Flag-RagD S77L (Addgene #19317) were gifts from David Sabatini.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot, Control, Immunostaining, Staining, Quantitative RT-PCR