plko 1 p18 shrna Search Results


lamtor1  (Addgene inc)
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Addgene inc lamtor1
( a ) HeLa cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 μM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. ( b ) Endogenous SLC38A9 was knocked down by lentivirus-transduced shRNAs as assessed by RT-qPCR. ( c ) The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. SLC38A9 knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for p-S6K1 and GAPDH. ( d ) SLC38A9 is required for the AA-simulated Golgi trafficking. Cells knocked down by indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a . ( e ) Immuno-blots showing that endogenous <t>Lamtor1</t> and RagA/B were knocked down by respective lentivirus-transduced shRNAs. ( f ) Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was conducted similarly to d . ( g ) mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin or 250 nM Torin1 was present throughout the treatment. ( h ) The immuno-blot showing that endogenous Arl5b was knocked down by a mixture of siRNAs targeting Arl5a, b and c. ( i ) Arl5 is required for the AA-stimulated Golgi trafficking of CD8a-furin. Experiment was conducted similarly to d . ( j ) The knockdown of endogenous Vps51 and Vps54 by respective lentivirus-transduced shRNAs as assessed by RT-qPCR. ( k ) GARP is required for the AA-stimulated Golgi trafficking. Experiment was conducted similarly to d . In a , d , f , g , I and k , the displayed value is the mean of n=3 independent experiments with each experiment analyzing ≥ 90, 21, 61, 50, 51 and 31 cells respectively; error bar, s.d.. P -values were from t -test. N.S., not significant ( P > 0.05); *, P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA.
Lamtor1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( a ) HeLa cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 μM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. ( b ) Endogenous SLC38A9 was knocked down by lentivirus-transduced shRNAs as assessed by RT-qPCR. ( c ) The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. SLC38A9 knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for p-S6K1 and GAPDH. ( d ) SLC38A9 is required for the AA-simulated Golgi trafficking. Cells knocked down by indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a . ( e ) Immuno-blots showing that endogenous Lamtor1 and RagA/B were knocked down by respective lentivirus-transduced shRNAs. ( f ) Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was conducted similarly to d . ( g ) mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin or 250 nM Torin1 was present throughout the treatment. ( h ) The immuno-blot showing that endogenous Arl5b was knocked down by a mixture of siRNAs targeting Arl5a, b and c. ( i ) Arl5 is required for the AA-stimulated Golgi trafficking of CD8a-furin. Experiment was conducted similarly to d . ( j ) The knockdown of endogenous Vps51 and Vps54 by respective lentivirus-transduced shRNAs as assessed by RT-qPCR. ( k ) GARP is required for the AA-stimulated Golgi trafficking. Experiment was conducted similarly to d . In a , d , f , g , I and k , the displayed value is the mean of n=3 independent experiments with each experiment analyzing ≥ 90, 21, 61, 50, 51 and 31 cells respectively; error bar, s.d.. P -values were from t -test. N.S., not significant ( P > 0.05); *, P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA.

Journal: bioRxiv

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1101/312546

Figure Lengend Snippet: ( a ) HeLa cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 μM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. ( b ) Endogenous SLC38A9 was knocked down by lentivirus-transduced shRNAs as assessed by RT-qPCR. ( c ) The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. SLC38A9 knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for p-S6K1 and GAPDH. ( d ) SLC38A9 is required for the AA-simulated Golgi trafficking. Cells knocked down by indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a . ( e ) Immuno-blots showing that endogenous Lamtor1 and RagA/B were knocked down by respective lentivirus-transduced shRNAs. ( f ) Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was conducted similarly to d . ( g ) mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin or 250 nM Torin1 was present throughout the treatment. ( h ) The immuno-blot showing that endogenous Arl5b was knocked down by a mixture of siRNAs targeting Arl5a, b and c. ( i ) Arl5 is required for the AA-stimulated Golgi trafficking of CD8a-furin. Experiment was conducted similarly to d . ( j ) The knockdown of endogenous Vps51 and Vps54 by respective lentivirus-transduced shRNAs as assessed by RT-qPCR. ( k ) GARP is required for the AA-stimulated Golgi trafficking. Experiment was conducted similarly to d . In a , d , f , g , I and k , the displayed value is the mean of n=3 independent experiments with each experiment analyzing ≥ 90, 21, 61, 50, 51 and 31 cells respectively; error bar, s.d.. P -values were from t -test. N.S., not significant ( P > 0.05); *, P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA.

Article Snippet: Lamtor1 (Addgene: #26631), Lamtor3 (Addgene: #26632), RagA #1 (Addgene: #30319), RagB #1 (Addgene: #26627).

Techniques: Stable Transfection, Expressing, Labeling, Incubation, Staining, Imaging, Quantitative RT-PCR, Knockdown, Activity Assay, Transfection, Western Blot, Control, shRNA

HEK293T cells were used. ( a ) Arl5b, but not Arl1, specifically pulled down Lamtor1-GFP. Bead-immobilized GST-Arl5b or Arl1 was in vitro loaded with GDP or GMPPNP and subsequently incubated with cell lysates expressing GFP or Lamtor1-GFP. Pull-downs were analyzed by immuno-blotting GFP-fusions. The loading of GST-fusions were shown by Coomassie blue staining. “1” and “2” indicate Lamtor1-GFP and GFP band, respectively. ( b,c ) Arl5b-QL and-TN interact with Lamtor1-Myc in forward and reverse co-IPs. Cells co-expressing indicated tagged-proteins were incubated with indicated antibodies and IPs were immuno-blotted against indicated tags. Lamtor2-GFP and SNX3-Myc served as a positive and negative control, respectively. In b , “1” and “2” indicate Arl5b-(wt, QL or TN)-GFP and Lamtor2-GFP band, respectively. In c , “1-5” indicate IgG heavy chain, Arl5b-(wt, QL or TN)-GFP, Lamtor2-GFP, SNX3-Myc and Lamtor1-Myc band, respectively. (d) Full length Lamtor1 is required for Arl5b-Lamtor1 interaction. Bead-immobilized GST-Arl5b-TN was incubated with cell lysates expressing indicated fragments of Lamtor1 and pull-downs were analyzed by immuno-blotting GFP-fusions. “*” denotes the specific band. ( e ) Arl5-QL and-TN interact with Lamtor1 but not Lamtor2-5. Bead-immobilized GST-fusion was incubated with cell lysates expressing indicated Myc-tagged Lamtors and pull-downs were analyzed by immuno-blotting Myc-tagged proteins. Lamtor1-Str.-Myc, Lamtor1-G2A-Strep-Myc. ( f ) Arl5b-TN interacts with Ragulator through Lamtor1. Bead-immobilized GST-Arl5b-TN was incubated with cell lysates expressing indicated Lamtors and pull-downs were analyzed by immuno-blotting indicated tags. “1-4” indicate DMyc-Lamtor5, DMyc-Lamtor4, GST-Arl5b-TN and GST band, respectively. ( g ) Arl5b-GMPPNP,-GDP and guanine nucleotide empty form interact with Ragulator. Bead-immobilized GST-Arl5b were first loaded with GMPPNP or GDP or stripped off its bound guanine nucleotide by EDTA treatment. Beads were subsequently incubated with cell lysate and pull-downs were analyzed by immuno-blotting endogenous Lamtor1-4.

Journal: bioRxiv

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1101/312546

Figure Lengend Snippet: HEK293T cells were used. ( a ) Arl5b, but not Arl1, specifically pulled down Lamtor1-GFP. Bead-immobilized GST-Arl5b or Arl1 was in vitro loaded with GDP or GMPPNP and subsequently incubated with cell lysates expressing GFP or Lamtor1-GFP. Pull-downs were analyzed by immuno-blotting GFP-fusions. The loading of GST-fusions were shown by Coomassie blue staining. “1” and “2” indicate Lamtor1-GFP and GFP band, respectively. ( b,c ) Arl5b-QL and-TN interact with Lamtor1-Myc in forward and reverse co-IPs. Cells co-expressing indicated tagged-proteins were incubated with indicated antibodies and IPs were immuno-blotted against indicated tags. Lamtor2-GFP and SNX3-Myc served as a positive and negative control, respectively. In b , “1” and “2” indicate Arl5b-(wt, QL or TN)-GFP and Lamtor2-GFP band, respectively. In c , “1-5” indicate IgG heavy chain, Arl5b-(wt, QL or TN)-GFP, Lamtor2-GFP, SNX3-Myc and Lamtor1-Myc band, respectively. (d) Full length Lamtor1 is required for Arl5b-Lamtor1 interaction. Bead-immobilized GST-Arl5b-TN was incubated with cell lysates expressing indicated fragments of Lamtor1 and pull-downs were analyzed by immuno-blotting GFP-fusions. “*” denotes the specific band. ( e ) Arl5-QL and-TN interact with Lamtor1 but not Lamtor2-5. Bead-immobilized GST-fusion was incubated with cell lysates expressing indicated Myc-tagged Lamtors and pull-downs were analyzed by immuno-blotting Myc-tagged proteins. Lamtor1-Str.-Myc, Lamtor1-G2A-Strep-Myc. ( f ) Arl5b-TN interacts with Ragulator through Lamtor1. Bead-immobilized GST-Arl5b-TN was incubated with cell lysates expressing indicated Lamtors and pull-downs were analyzed by immuno-blotting indicated tags. “1-4” indicate DMyc-Lamtor5, DMyc-Lamtor4, GST-Arl5b-TN and GST band, respectively. ( g ) Arl5b-GMPPNP,-GDP and guanine nucleotide empty form interact with Ragulator. Bead-immobilized GST-Arl5b were first loaded with GMPPNP or GDP or stripped off its bound guanine nucleotide by EDTA treatment. Beads were subsequently incubated with cell lysate and pull-downs were analyzed by immuno-blotting endogenous Lamtor1-4.

Article Snippet: Lamtor1 (Addgene: #26631), Lamtor3 (Addgene: #26632), RagA #1 (Addgene: #30319), RagB #1 (Addgene: #26627).

Techniques: In Vitro, Incubation, Expressing, Staining, Negative Control

Arl5a and b are highly similar in primary sequence and both interact with Lamtor1. ( a ) Multiple sequence alignment of human and mouse Arl5a, b and c. The GenBank Accession number of each protein sequence is indicated. The five highly conserved guanine nucleotide binding motifs, G1-5 boxes, are underlined. The protein sequence of hArl5c is significantly different from that of Arl5a, Arl5b or mArl5c, especially at G3. The multiple alignment was conducted in Vector NTI (Invitrogen). ( b ) Human Arl5a and b and mouse Arl5c interact with Lamtor1. Bead-immobilized GST-fusions were incubated with cell lysate expressing Lamtor1-GFP and pull-downs were analyzed by immuno-blotting GFP-fusions. The prefix h and m denote human and mouse, respectively.

Journal: bioRxiv

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1101/312546

Figure Lengend Snippet: Arl5a and b are highly similar in primary sequence and both interact with Lamtor1. ( a ) Multiple sequence alignment of human and mouse Arl5a, b and c. The GenBank Accession number of each protein sequence is indicated. The five highly conserved guanine nucleotide binding motifs, G1-5 boxes, are underlined. The protein sequence of hArl5c is significantly different from that of Arl5a, Arl5b or mArl5c, especially at G3. The multiple alignment was conducted in Vector NTI (Invitrogen). ( b ) Human Arl5a and b and mouse Arl5c interact with Lamtor1. Bead-immobilized GST-fusions were incubated with cell lysate expressing Lamtor1-GFP and pull-downs were analyzed by immuno-blotting GFP-fusions. The prefix h and m denote human and mouse, respectively.

Article Snippet: Lamtor1 (Addgene: #26631), Lamtor3 (Addgene: #26632), RagA #1 (Addgene: #30319), RagB #1 (Addgene: #26627).

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Incubation, Expressing

All cells are HeLa cells. ( a ) The Golgi localization of different mutant forms of Arl5b. Cells transiently expressing Arl5b-GFP in QL, TN or wt form were fixed and endogenous Golgin-245 was stained. ( b ) Endogenous Arl5b localizes to the Golgi. HeLa cells were fixed and endogenous Arl5b and GS28 were co-stained. ( c,d ) Arl5b colocalizes with Lamtor1 at the endosome and lysosome. HeLa cells transiently co-expressing indicated GFP or mCherry-tagged proteins were imaged under live cell condition. ( e ) Lamtor1 localizes to the EE, LE and lysosome. Endogenous Lamtor1 was co-stained with exogenously expressed GFP-Rab7, endogenous EEA1 or Lamp1, respectively. In c-e , the boxed region was enlarged in the upper right corner to show the colocalization at puncta (denoted by arrows). Scale bar, 10 μm.

Journal: bioRxiv

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1101/312546

Figure Lengend Snippet: All cells are HeLa cells. ( a ) The Golgi localization of different mutant forms of Arl5b. Cells transiently expressing Arl5b-GFP in QL, TN or wt form were fixed and endogenous Golgin-245 was stained. ( b ) Endogenous Arl5b localizes to the Golgi. HeLa cells were fixed and endogenous Arl5b and GS28 were co-stained. ( c,d ) Arl5b colocalizes with Lamtor1 at the endosome and lysosome. HeLa cells transiently co-expressing indicated GFP or mCherry-tagged proteins were imaged under live cell condition. ( e ) Lamtor1 localizes to the EE, LE and lysosome. Endogenous Lamtor1 was co-stained with exogenously expressed GFP-Rab7, endogenous EEA1 or Lamp1, respectively. In c-e , the boxed region was enlarged in the upper right corner to show the colocalization at puncta (denoted by arrows). Scale bar, 10 μm.

Article Snippet: Lamtor1 (Addgene: #26631), Lamtor3 (Addgene: #26632), RagA #1 (Addgene: #30319), RagB #1 (Addgene: #26627).

Techniques: Mutagenesis, Expressing, Staining

This figure corresponds to . ( a,b,c ) Arl5a and mArl5c, but not hArl5c, localize to the Golgi. HeLa cells transiently expressing indicated GFP-fusions were fixed and endogenous Golgin-245 or giantin was stained. The prefix h and m denote human and mouse, respectively. By default, Arl5 paralogs are from human. ( d,e,f ) Characterization of anti-Arl5b rabbit pAb. In d , HeLa cell lysates were subjected to co-IP by indicated antibodies and co-IPs were immuno-blotted by anti-Arl5b rabbit pAb. In e , HeLa cell lysates transiently expressing Arl5b-GFP were subjected to co-IP by indicated antibodies and co-IPs were analyzed by immuno-blotting GFP-fusions. To prepare Arl5b-depleted pAb, Arl5b pAb was incubated with sufficient amount of bead-immobilized GST-Arl5b and the supernatant was saved. In f , HeLa cell lysate transiently expressing GFP-tagged Arl5a, Arl5b or mArl5c was subjected to immuno-blotting by anti-GFP mAb and anti-Arl5b pAb, demonstrating that our anti-Arl5b pAb preferentially recognizes Arl5b. ( g ) N-terminal myristoylation is probably required for the Golgi localization of Arl5b. HeLa cells transiently expressing Arl5b-QL-GFP or Arl5b-QL-GFP harboring G2A mutation were fixed and endogenous Golgin-245 was stained. In Arl5b, Gly at position 2 is a potential myristoylation site. ( h ) Arl5b-wt does not significantly localize to the endosome or lysosome. Live imaging of HeLa cells expressing indicated proteins. ( i ) Lamtor1 does not localize to the Golgi. HeLa cells were stained for endogenous Lamtor1 and Golgin-245. Scale bar, 10 μm.

Journal: bioRxiv

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1101/312546

Figure Lengend Snippet: This figure corresponds to . ( a,b,c ) Arl5a and mArl5c, but not hArl5c, localize to the Golgi. HeLa cells transiently expressing indicated GFP-fusions were fixed and endogenous Golgin-245 or giantin was stained. The prefix h and m denote human and mouse, respectively. By default, Arl5 paralogs are from human. ( d,e,f ) Characterization of anti-Arl5b rabbit pAb. In d , HeLa cell lysates were subjected to co-IP by indicated antibodies and co-IPs were immuno-blotted by anti-Arl5b rabbit pAb. In e , HeLa cell lysates transiently expressing Arl5b-GFP were subjected to co-IP by indicated antibodies and co-IPs were analyzed by immuno-blotting GFP-fusions. To prepare Arl5b-depleted pAb, Arl5b pAb was incubated with sufficient amount of bead-immobilized GST-Arl5b and the supernatant was saved. In f , HeLa cell lysate transiently expressing GFP-tagged Arl5a, Arl5b or mArl5c was subjected to immuno-blotting by anti-GFP mAb and anti-Arl5b pAb, demonstrating that our anti-Arl5b pAb preferentially recognizes Arl5b. ( g ) N-terminal myristoylation is probably required for the Golgi localization of Arl5b. HeLa cells transiently expressing Arl5b-QL-GFP or Arl5b-QL-GFP harboring G2A mutation were fixed and endogenous Golgin-245 was stained. In Arl5b, Gly at position 2 is a potential myristoylation site. ( h ) Arl5b-wt does not significantly localize to the endosome or lysosome. Live imaging of HeLa cells expressing indicated proteins. ( i ) Lamtor1 does not localize to the Golgi. HeLa cells were stained for endogenous Lamtor1 and Golgin-245. Scale bar, 10 μm.

Article Snippet: Lamtor1 (Addgene: #26631), Lamtor3 (Addgene: #26632), RagA #1 (Addgene: #30319), RagB #1 (Addgene: #26627).

Techniques: Expressing, Staining, Co-Immunoprecipitation Assay, Incubation, Mutagenesis, Imaging