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  • 91
    Addgene inc prk5 ha c7orf59 lamtor4
    (A) p27 was immunoprecipitated from HEK293 cells transfected with p27 or p27 CK− and/or HA-LAMTOR1 (L1), membranes were probed with anti-HA (LAMTOR1) and p27. Transfected protein levels were determined in extracts with the same antibodies. Grb2 was used as loading control. (B) Pull-downs of recombinant His-tagged LAMTOR1 with GST-p27 or GST-p27 CK -beads, GST only beads were used as control. The amounts of beads used were determined by Coomassie staining. A tenth of His-LAMTOR1 input at the same exposure time is shown on the right. (C) HEK293 expressing Flag-LAMTOR1 were subjected to pull-downs using GST beads of various p27 deletion mutants and immunoblotted with anti-Flag (LAMTOR1) antibodies. p27 N T = aa 1-87 and p27 C T = aa 88-198. The amounts of beads used were determined by Coomassie staining. Ectopic expression of LAMTOR1 in cell extract was confirmed by anti-Flag immunoblot. (D) Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates with anti-LAMTOR1 antibodies and blotted against p27 and LAMTOR1. Protein A beads were used as negative control. Levels of endogenous proteins were determined with anti-p27 and anti-LAMTOR1 antibody. (E) PLA using p27 and LAMTOR1 antibodies on p27 +/+ MEFs in full medium or aa deprived for 18 h. p27 −/− MEFs were used as negative control. F-actin was stained with phalloïdin. Scale bar are 50 µm. Controls PLA reactions with single antibody or without primary antibodies are shown in Fig. S2. (F) Bar graph shows the mean number of PLA dots per cell ± SEM as described in E (n cells counted: p27 +/+ 0 h=2087, p27 +/+ 18 h=1677, p27 −/− 0 h=579, p27 −/− 18 h=225). Statistical significance was evaluated by 2-way ANOVA; **: p ≤ 0.01. (G) HEK293 cells were transfected with tagged LAMTOR1 and/or another Ragulator subunit and/or p27. LAMTOR1 co-IPs were probed for the co-transfected Ragulator subunit to determine the impact of p27 expression on the ability of LAMTOR1 to interact with its partners. (H) Densitometry analysis of experiments described in G. Signal intensity values of LAMTOR2, −3, − 4 and −5 bound to LAMTOR1 were normalized to that in absence of p27. Bar graph shows means ± SEM from 3 independent experiments. Statistical significance was evaluated by 2-way ANOVA; ns: p> 0.05; ****: p ≤ 0.0001. (I) p27 was immunoprecipitated from p27 +/+ MEFs in full medium or aa starved for 24 h, p27 −/− cells aa starved for 24 h were used as negative control. The amount of <t>LAMTOR4</t> co-precipitated with p27 was determined. Levels of LAMTOR4 and p27 in the corresponding extracts are shown. β-actin was used as loading control. (J) LAMTOR1 was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR5 co-precipitated was determined. Control immunoprecipitation with rabbit IgG was used as control. Levels of LAMTOR1 and −5 in the corresponding extracts are shown. β-actin and β-tubulin were used as loading control. (K) Ratio of LAMTOR5 co-precipitated by that of LAMTOR1 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 J. (L) Schematic summarizing the impact of p27 on Ragulator assembly.
    Prk5 Ha C7orf59 Lamtor4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    NCIMB Ltd accession no 42336
    (A) p27 was immunoprecipitated from HEK293 cells transfected with p27 or p27 CK− and/or HA-LAMTOR1 (L1), membranes were probed with anti-HA (LAMTOR1) and p27. Transfected protein levels were determined in extracts with the same antibodies. Grb2 was used as loading control. (B) Pull-downs of recombinant His-tagged LAMTOR1 with GST-p27 or GST-p27 CK -beads, GST only beads were used as control. The amounts of beads used were determined by Coomassie staining. A tenth of His-LAMTOR1 input at the same exposure time is shown on the right. (C) HEK293 expressing Flag-LAMTOR1 were subjected to pull-downs using GST beads of various p27 deletion mutants and immunoblotted with anti-Flag (LAMTOR1) antibodies. p27 N T = aa 1-87 and p27 C T = aa 88-198. The amounts of beads used were determined by Coomassie staining. Ectopic expression of LAMTOR1 in cell extract was confirmed by anti-Flag immunoblot. (D) Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates with anti-LAMTOR1 antibodies and blotted against p27 and LAMTOR1. Protein A beads were used as negative control. Levels of endogenous proteins were determined with anti-p27 and anti-LAMTOR1 antibody. (E) PLA using p27 and LAMTOR1 antibodies on p27 +/+ MEFs in full medium or aa deprived for 18 h. p27 −/− MEFs were used as negative control. F-actin was stained with phalloïdin. Scale bar are 50 µm. Controls PLA reactions with single antibody or without primary antibodies are shown in Fig. S2. (F) Bar graph shows the mean number of PLA dots per cell ± SEM as described in E (n cells counted: p27 +/+ 0 h=2087, p27 +/+ 18 h=1677, p27 −/− 0 h=579, p27 −/− 18 h=225). Statistical significance was evaluated by 2-way ANOVA; **: p ≤ 0.01. (G) HEK293 cells were transfected with tagged LAMTOR1 and/or another Ragulator subunit and/or p27. LAMTOR1 co-IPs were probed for the co-transfected Ragulator subunit to determine the impact of p27 expression on the ability of LAMTOR1 to interact with its partners. (H) Densitometry analysis of experiments described in G. Signal intensity values of LAMTOR2, −3, − 4 and −5 bound to LAMTOR1 were normalized to that in absence of p27. Bar graph shows means ± SEM from 3 independent experiments. Statistical significance was evaluated by 2-way ANOVA; ns: p> 0.05; ****: p ≤ 0.0001. (I) p27 was immunoprecipitated from p27 +/+ MEFs in full medium or aa starved for 24 h, p27 −/− cells aa starved for 24 h were used as negative control. The amount of <t>LAMTOR4</t> co-precipitated with p27 was determined. Levels of LAMTOR4 and p27 in the corresponding extracts are shown. β-actin was used as loading control. (J) LAMTOR1 was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR5 co-precipitated was determined. Control immunoprecipitation with rabbit IgG was used as control. Levels of LAMTOR1 and −5 in the corresponding extracts are shown. β-actin and β-tubulin were used as loading control. (K) Ratio of LAMTOR5 co-precipitated by that of LAMTOR1 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 J. (L) Schematic summarizing the impact of p27 on Ragulator assembly.
    Accession No 42336, supplied by NCIMB Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    accession no 42336 - by Bioz Stars, 2024-02
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    86
    Icare Inc nt05 3 42336
    (A) p27 was immunoprecipitated from HEK293 cells transfected with p27 or p27 CK− and/or HA-LAMTOR1 (L1), membranes were probed with anti-HA (LAMTOR1) and p27. Transfected protein levels were determined in extracts with the same antibodies. Grb2 was used as loading control. (B) Pull-downs of recombinant His-tagged LAMTOR1 with GST-p27 or GST-p27 CK -beads, GST only beads were used as control. The amounts of beads used were determined by Coomassie staining. A tenth of His-LAMTOR1 input at the same exposure time is shown on the right. (C) HEK293 expressing Flag-LAMTOR1 were subjected to pull-downs using GST beads of various p27 deletion mutants and immunoblotted with anti-Flag (LAMTOR1) antibodies. p27 N T = aa 1-87 and p27 C T = aa 88-198. The amounts of beads used were determined by Coomassie staining. Ectopic expression of LAMTOR1 in cell extract was confirmed by anti-Flag immunoblot. (D) Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates with anti-LAMTOR1 antibodies and blotted against p27 and LAMTOR1. Protein A beads were used as negative control. Levels of endogenous proteins were determined with anti-p27 and anti-LAMTOR1 antibody. (E) PLA using p27 and LAMTOR1 antibodies on p27 +/+ MEFs in full medium or aa deprived for 18 h. p27 −/− MEFs were used as negative control. F-actin was stained with phalloïdin. Scale bar are 50 µm. Controls PLA reactions with single antibody or without primary antibodies are shown in Fig. S2. (F) Bar graph shows the mean number of PLA dots per cell ± SEM as described in E (n cells counted: p27 +/+ 0 h=2087, p27 +/+ 18 h=1677, p27 −/− 0 h=579, p27 −/− 18 h=225). Statistical significance was evaluated by 2-way ANOVA; **: p ≤ 0.01. (G) HEK293 cells were transfected with tagged LAMTOR1 and/or another Ragulator subunit and/or p27. LAMTOR1 co-IPs were probed for the co-transfected Ragulator subunit to determine the impact of p27 expression on the ability of LAMTOR1 to interact with its partners. (H) Densitometry analysis of experiments described in G. Signal intensity values of LAMTOR2, −3, − 4 and −5 bound to LAMTOR1 were normalized to that in absence of p27. Bar graph shows means ± SEM from 3 independent experiments. Statistical significance was evaluated by 2-way ANOVA; ns: p> 0.05; ****: p ≤ 0.0001. (I) p27 was immunoprecipitated from p27 +/+ MEFs in full medium or aa starved for 24 h, p27 −/− cells aa starved for 24 h were used as negative control. The amount of <t>LAMTOR4</t> co-precipitated with p27 was determined. Levels of LAMTOR4 and p27 in the corresponding extracts are shown. β-actin was used as loading control. (J) LAMTOR1 was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR5 co-precipitated was determined. Control immunoprecipitation with rabbit IgG was used as control. Levels of LAMTOR1 and −5 in the corresponding extracts are shown. β-actin and β-tubulin were used as loading control. (K) Ratio of LAMTOR5 co-precipitated by that of LAMTOR1 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 J. (L) Schematic summarizing the impact of p27 on Ragulator assembly.
    Nt05 3 42336, supplied by Icare Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    NCIMB Ltd ncimb accession no 42336
    (A) p27 was immunoprecipitated from HEK293 cells transfected with p27 or p27 CK− and/or HA-LAMTOR1 (L1), membranes were probed with anti-HA (LAMTOR1) and p27. Transfected protein levels were determined in extracts with the same antibodies. Grb2 was used as loading control. (B) Pull-downs of recombinant His-tagged LAMTOR1 with GST-p27 or GST-p27 CK -beads, GST only beads were used as control. The amounts of beads used were determined by Coomassie staining. A tenth of His-LAMTOR1 input at the same exposure time is shown on the right. (C) HEK293 expressing Flag-LAMTOR1 were subjected to pull-downs using GST beads of various p27 deletion mutants and immunoblotted with anti-Flag (LAMTOR1) antibodies. p27 N T = aa 1-87 and p27 C T = aa 88-198. The amounts of beads used were determined by Coomassie staining. Ectopic expression of LAMTOR1 in cell extract was confirmed by anti-Flag immunoblot. (D) Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates with anti-LAMTOR1 antibodies and blotted against p27 and LAMTOR1. Protein A beads were used as negative control. Levels of endogenous proteins were determined with anti-p27 and anti-LAMTOR1 antibody. (E) PLA using p27 and LAMTOR1 antibodies on p27 +/+ MEFs in full medium or aa deprived for 18 h. p27 −/− MEFs were used as negative control. F-actin was stained with phalloïdin. Scale bar are 50 µm. Controls PLA reactions with single antibody or without primary antibodies are shown in Fig. S2. (F) Bar graph shows the mean number of PLA dots per cell ± SEM as described in E (n cells counted: p27 +/+ 0 h=2087, p27 +/+ 18 h=1677, p27 −/− 0 h=579, p27 −/− 18 h=225). Statistical significance was evaluated by 2-way ANOVA; **: p ≤ 0.01. (G) HEK293 cells were transfected with tagged LAMTOR1 and/or another Ragulator subunit and/or p27. LAMTOR1 co-IPs were probed for the co-transfected Ragulator subunit to determine the impact of p27 expression on the ability of LAMTOR1 to interact with its partners. (H) Densitometry analysis of experiments described in G. Signal intensity values of LAMTOR2, −3, − 4 and −5 bound to LAMTOR1 were normalized to that in absence of p27. Bar graph shows means ± SEM from 3 independent experiments. Statistical significance was evaluated by 2-way ANOVA; ns: p> 0.05; ****: p ≤ 0.0001. (I) p27 was immunoprecipitated from p27 +/+ MEFs in full medium or aa starved for 24 h, p27 −/− cells aa starved for 24 h were used as negative control. The amount of <t>LAMTOR4</t> co-precipitated with p27 was determined. Levels of LAMTOR4 and p27 in the corresponding extracts are shown. β-actin was used as loading control. (J) LAMTOR1 was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR5 co-precipitated was determined. Control immunoprecipitation with rabbit IgG was used as control. Levels of LAMTOR1 and −5 in the corresponding extracts are shown. β-actin and β-tubulin were used as loading control. (K) Ratio of LAMTOR5 co-precipitated by that of LAMTOR1 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 J. (L) Schematic summarizing the impact of p27 on Ragulator assembly.
    Ncimb Accession No 42336, supplied by NCIMB Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ncimb accession no 42336 - by Bioz Stars, 2024-02
    86/100 stars
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    Image Search Results


    (A) p27 was immunoprecipitated from HEK293 cells transfected with p27 or p27 CK− and/or HA-LAMTOR1 (L1), membranes were probed with anti-HA (LAMTOR1) and p27. Transfected protein levels were determined in extracts with the same antibodies. Grb2 was used as loading control. (B) Pull-downs of recombinant His-tagged LAMTOR1 with GST-p27 or GST-p27 CK -beads, GST only beads were used as control. The amounts of beads used were determined by Coomassie staining. A tenth of His-LAMTOR1 input at the same exposure time is shown on the right. (C) HEK293 expressing Flag-LAMTOR1 were subjected to pull-downs using GST beads of various p27 deletion mutants and immunoblotted with anti-Flag (LAMTOR1) antibodies. p27 N T = aa 1-87 and p27 C T = aa 88-198. The amounts of beads used were determined by Coomassie staining. Ectopic expression of LAMTOR1 in cell extract was confirmed by anti-Flag immunoblot. (D) Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates with anti-LAMTOR1 antibodies and blotted against p27 and LAMTOR1. Protein A beads were used as negative control. Levels of endogenous proteins were determined with anti-p27 and anti-LAMTOR1 antibody. (E) PLA using p27 and LAMTOR1 antibodies on p27 +/+ MEFs in full medium or aa deprived for 18 h. p27 −/− MEFs were used as negative control. F-actin was stained with phalloïdin. Scale bar are 50 µm. Controls PLA reactions with single antibody or without primary antibodies are shown in Fig. S2. (F) Bar graph shows the mean number of PLA dots per cell ± SEM as described in E (n cells counted: p27 +/+ 0 h=2087, p27 +/+ 18 h=1677, p27 −/− 0 h=579, p27 −/− 18 h=225). Statistical significance was evaluated by 2-way ANOVA; **: p ≤ 0.01. (G) HEK293 cells were transfected with tagged LAMTOR1 and/or another Ragulator subunit and/or p27. LAMTOR1 co-IPs were probed for the co-transfected Ragulator subunit to determine the impact of p27 expression on the ability of LAMTOR1 to interact with its partners. (H) Densitometry analysis of experiments described in G. Signal intensity values of LAMTOR2, −3, − 4 and −5 bound to LAMTOR1 were normalized to that in absence of p27. Bar graph shows means ± SEM from 3 independent experiments. Statistical significance was evaluated by 2-way ANOVA; ns: p> 0.05; ****: p ≤ 0.0001. (I) p27 was immunoprecipitated from p27 +/+ MEFs in full medium or aa starved for 24 h, p27 −/− cells aa starved for 24 h were used as negative control. The amount of LAMTOR4 co-precipitated with p27 was determined. Levels of LAMTOR4 and p27 in the corresponding extracts are shown. β-actin was used as loading control. (J) LAMTOR1 was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR5 co-precipitated was determined. Control immunoprecipitation with rabbit IgG was used as control. Levels of LAMTOR1 and −5 in the corresponding extracts are shown. β-actin and β-tubulin were used as loading control. (K) Ratio of LAMTOR5 co-precipitated by that of LAMTOR1 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 J. (L) Schematic summarizing the impact of p27 on Ragulator assembly.

    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) p27 was immunoprecipitated from HEK293 cells transfected with p27 or p27 CK− and/or HA-LAMTOR1 (L1), membranes were probed with anti-HA (LAMTOR1) and p27. Transfected protein levels were determined in extracts with the same antibodies. Grb2 was used as loading control. (B) Pull-downs of recombinant His-tagged LAMTOR1 with GST-p27 or GST-p27 CK -beads, GST only beads were used as control. The amounts of beads used were determined by Coomassie staining. A tenth of His-LAMTOR1 input at the same exposure time is shown on the right. (C) HEK293 expressing Flag-LAMTOR1 were subjected to pull-downs using GST beads of various p27 deletion mutants and immunoblotted with anti-Flag (LAMTOR1) antibodies. p27 N T = aa 1-87 and p27 C T = aa 88-198. The amounts of beads used were determined by Coomassie staining. Ectopic expression of LAMTOR1 in cell extract was confirmed by anti-Flag immunoblot. (D) Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates with anti-LAMTOR1 antibodies and blotted against p27 and LAMTOR1. Protein A beads were used as negative control. Levels of endogenous proteins were determined with anti-p27 and anti-LAMTOR1 antibody. (E) PLA using p27 and LAMTOR1 antibodies on p27 +/+ MEFs in full medium or aa deprived for 18 h. p27 −/− MEFs were used as negative control. F-actin was stained with phalloïdin. Scale bar are 50 µm. Controls PLA reactions with single antibody or without primary antibodies are shown in Fig. S2. (F) Bar graph shows the mean number of PLA dots per cell ± SEM as described in E (n cells counted: p27 +/+ 0 h=2087, p27 +/+ 18 h=1677, p27 −/− 0 h=579, p27 −/− 18 h=225). Statistical significance was evaluated by 2-way ANOVA; **: p ≤ 0.01. (G) HEK293 cells were transfected with tagged LAMTOR1 and/or another Ragulator subunit and/or p27. LAMTOR1 co-IPs were probed for the co-transfected Ragulator subunit to determine the impact of p27 expression on the ability of LAMTOR1 to interact with its partners. (H) Densitometry analysis of experiments described in G. Signal intensity values of LAMTOR2, −3, − 4 and −5 bound to LAMTOR1 were normalized to that in absence of p27. Bar graph shows means ± SEM from 3 independent experiments. Statistical significance was evaluated by 2-way ANOVA; ns: p> 0.05; ****: p ≤ 0.0001. (I) p27 was immunoprecipitated from p27 +/+ MEFs in full medium or aa starved for 24 h, p27 −/− cells aa starved for 24 h were used as negative control. The amount of LAMTOR4 co-precipitated with p27 was determined. Levels of LAMTOR4 and p27 in the corresponding extracts are shown. β-actin was used as loading control. (J) LAMTOR1 was immunoprecipitated from p27 +/+ and p27 −/− MEFs in full medium or aa starved for 24 h and the amount of LAMTOR5 co-precipitated was determined. Control immunoprecipitation with rabbit IgG was used as control. Levels of LAMTOR1 and −5 in the corresponding extracts are shown. β-actin and β-tubulin were used as loading control. (K) Ratio of LAMTOR5 co-precipitated by that of LAMTOR1 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 J. (L) Schematic summarizing the impact of p27 on Ragulator assembly.

    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, Transfection, Recombinant, Staining, Expressing, Western Blot, Negative Control

    (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.

    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, Immunostaining, Staining, Quantitative RT-PCR