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Proteintech rabbit anti mapksp1
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Rabbit Anti Mapksp1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+mapksp1/MAPKSP1+Antibody/pmc11876615-26-0-3
Average 93 stars, based on 1 article reviews

rabbit anti mapksp1 - by Bioz Stars, 2026-10

93/100 stars

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1) Product Images from "Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis"

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

Journal: The EMBO Journal

doi: 10.1038/s44318-024-00359-z

Figure Legend Snippet: Reagents and tools table

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

Recombinant:

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis
Article Snippet: Rabbit anti-MAPKSP1 , Proteintech, USA , Cat #11937-1-AP.

Sequencing:

Modification:

Membrane:

Lysis:

Transfection:

Magnetic Beads:

Software:

Cytometry:

In Vitro:

cDNA Synthesis:

Plasmid Preparation:

Isolation:

Mutagenesis:

Silver Staining:

Image Search Results

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: Rabbit anti-MAPKSP1 , Proteintech, USA , Cat #11937-1-AP.

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

Loss of Ragulator components blocks EV71-induced apoptosis and pyroptosis. (A) Schematic diagram of the genome-wide CRISPR screening process and Western blot analysis of Cas9 expression in HeLa cells. (B and C) Sequencing analysis of knockout efficiency of LAMTOR3 and LAMTOR1 . (D–F) LAMTOR2 and LAMTOR4 were silenced in HeLa cells using siRNAs and then the indicated HeLa cells were infected with EV71 (MOI = 5) 60 h after transfection. The knockdown efficiency of LAMTOR2 (D) and LAMTOR4 (E) was determined by qPCR 60 h after transfection. Cell death induced by EV71 was detected by measuring ATP levels (D and E) and PI staining at 18 h after infection (F). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: Loss of Ragulator components blocks EV71-induced apoptosis and pyroptosis. (A) Schematic diagram of the genome-wide CRISPR screening process and Western blot analysis of Cas9 expression in HeLa cells. (B and C) Sequencing analysis of knockout efficiency of LAMTOR3 and LAMTOR1 . (D–F) LAMTOR2 and LAMTOR4 were silenced in HeLa cells using siRNAs and then the indicated HeLa cells were infected with EV71 (MOI = 5) 60 h after transfection. The knockdown efficiency of LAMTOR2 (D) and LAMTOR4 (E) was determined by qPCR 60 h after transfection. Cell death induced by EV71 was detected by measuring ATP levels (D and E) and PI staining at 18 h after infection (F). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Article Snippet: Antibodies were used as follows: LAMTOR3 (Cat #8168; Cell Signaling Technology), LAMTOR1 (Cat #8975S; Cell Signaling Technology), RagB (Cat #8150S, RRID:AB_11178806; Cell Signaling Technology), Enterovirus 71 (VP1; Cat #ab169442; Abcam), Enterovirus 71 3D (Cat #GTX630193, RRID:AB_2888196; GeneTex), GSDME (Cat #ab215191; Abcam), Cas9 (Cat #ab191468; Abcam), Caspase-3 (Cat #9662s; Cell Signaling Technology), Cleaved-Caspase-3 (Cat #9661; Cell Signaling Technology), FLAG-tag (Cat #A8592; Sigma-Aldrich), Caspase-9 (Cat #9508, RRID:AB_2068620; Cell Signaling Technology), Myc-tag (Cat #16286-1-AP, RRID:AB_11182162; Proteintech), β-actin (Cat #A2066; Sigma-Aldrich) anti-mouse IgG secondary antibody (Cat #92632210; LI-COR Biosciences), and anti-rabbit IgG secondary antibody (Cat #926-32211, RRID:AB_621843; LI-COR Biosciences).

Techniques: Genome Wide, CRISPR, Western Blot, Expressing, Sequencing, Knock-Out, Infection, Transfection, Knockdown, Staining, Two Tailed Test

Loss of Ragulator components blocks EV71-induced apoptosis and pyroptosis. (A) A genome-wide CRISPR screening identified a total of 13 genes in which more than three sgRNAs were expressed at least fivefold higher in EV71-infected cells compared to mock-infected cells (left panel). In addition to LAMTOR3 , other hits were identified that were either involved in enterovirus replication or acted as enterovirus receptors (right panel). The light red color represents genes that serve as receptors for enteroviruses. The blue color represents genes associated with enterovirus replication. The deep red color represents LAMTOR3 . (B) LAMTOR3 +/+ or LAMTOR3 −/− HeLa cells were infected (MOI = 5) with EV71 for 18 h and cell death was determined by measuring ATP levels (upper panel). The KO efficiency of LAMTOR3 in HeLa cells was assessed by Western blot analysis (lower panel). All values are means ± SEM. Data were analyzed using a two-tailed Student’s t test. (C) LAMTOR3 +/+ or LAMTOR3 −/− HeLa cells stably transfected with an empty vector ( LAMTOR3 −/− -1+Vector) or a vector expressing LAMTOR3 ( LAMTOR3 −/− -1 + LAMTOR3 ) were infected with EV71 (MOI = 5) for 18 h and cell death was determined by measuring ATP levels (upper panel). The rescue efficiency of ectopic expression of LAMTOR3 in LAMTOR3 -KO HeLa cells was analyzed by Western blotting (lower panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (D) HeLa cells as indicated ( LAMTOR3 +/+ , LAMTOR3 −/− -1, LAMTOR3 −/− -1+Vector, and LAMTOR3 −/− -1+ LAMTOR3 ) were infected with EV71 (MOI = 5) for 18 h and cell death was determined by PI staining. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (E and F) LAMTOR3 +/+ or LAMTOR3 −/− RD cells were infected with EV71 (MOI = 2) for 18 h and cell death was detected by measuring ATP levels (E) and PI staining (F). The knockout efficiency of LAMTOR3 in RD cells was determined by Western blot analysis (E). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G and H) LAMTOR1 +/+ or LAMTOR1 −/− HeLa cells were infected with EV71 (MOI = 5) for 18 h and cell death was detected by measuring ATP levels (G) and PI staining (H). The knockout efficiency of LAMTOR1 in HeLa cells was determined by Western blot analysis (G). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I and J) LAMTOR3 +/+ and LAMTOR3 −/− (I) or LAMTOR1 +/+ and LAMTOR1 −/− (J) HeLa cells were infected with EV71 (MOI = 5). Cleavage of GSDME and the activation of caspase3 and caspase9 were analyzed by Western blot 12 h after infection. The quantitation analysis of Western blot was performed using ImageJ. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: Loss of Ragulator components blocks EV71-induced apoptosis and pyroptosis. (A) A genome-wide CRISPR screening identified a total of 13 genes in which more than three sgRNAs were expressed at least fivefold higher in EV71-infected cells compared to mock-infected cells (left panel). In addition to LAMTOR3 , other hits were identified that were either involved in enterovirus replication or acted as enterovirus receptors (right panel). The light red color represents genes that serve as receptors for enteroviruses. The blue color represents genes associated with enterovirus replication. The deep red color represents LAMTOR3 . (B) LAMTOR3 +/+ or LAMTOR3 −/− HeLa cells were infected (MOI = 5) with EV71 for 18 h and cell death was determined by measuring ATP levels (upper panel). The KO efficiency of LAMTOR3 in HeLa cells was assessed by Western blot analysis (lower panel). All values are means ± SEM. Data were analyzed using a two-tailed Student’s t test. (C) LAMTOR3 +/+ or LAMTOR3 −/− HeLa cells stably transfected with an empty vector ( LAMTOR3 −/− -1+Vector) or a vector expressing LAMTOR3 ( LAMTOR3 −/− -1 + LAMTOR3 ) were infected with EV71 (MOI = 5) for 18 h and cell death was determined by measuring ATP levels (upper panel). The rescue efficiency of ectopic expression of LAMTOR3 in LAMTOR3 -KO HeLa cells was analyzed by Western blotting (lower panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (D) HeLa cells as indicated ( LAMTOR3 +/+ , LAMTOR3 −/− -1, LAMTOR3 −/− -1+Vector, and LAMTOR3 −/− -1+ LAMTOR3 ) were infected with EV71 (MOI = 5) for 18 h and cell death was determined by PI staining. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (E and F) LAMTOR3 +/+ or LAMTOR3 −/− RD cells were infected with EV71 (MOI = 2) for 18 h and cell death was detected by measuring ATP levels (E) and PI staining (F). The knockout efficiency of LAMTOR3 in RD cells was determined by Western blot analysis (E). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G and H) LAMTOR1 +/+ or LAMTOR1 −/− HeLa cells were infected with EV71 (MOI = 5) for 18 h and cell death was detected by measuring ATP levels (G) and PI staining (H). The knockout efficiency of LAMTOR1 in HeLa cells was determined by Western blot analysis (G). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I and J) LAMTOR3 +/+ and LAMTOR3 −/− (I) or LAMTOR1 +/+ and LAMTOR1 −/− (J) HeLa cells were infected with EV71 (MOI = 5). Cleavage of GSDME and the activation of caspase3 and caspase9 were analyzed by Western blot 12 h after infection. The quantitation analysis of Western blot was performed using ImageJ. Source data are available for this figure: .

Techniques: Genome Wide, CRISPR, Infection, Western Blot, Two Tailed Test, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Staining, Knock-Out, Activation Assay, Quantitation Assay

The Ragulator-Rag complex is required for EV71 replication. (A and B) Wild-type, LAMTOR1 −/− (A), and LAMTOR3 −/− (B) HeLa cells were infected with EV71 (MOI = 5) and the viral titers were determined by TCID50 assay at 12, 24, and 36 h after infection. (C–F) Wild-type, LAMTOR1 −/− , and LAMTOR3 −/− HeLa cells were incubated with EV71 (MOI > 100) at 4°C for 1 h. For the virus binding assay, cells were washed with 1×PBS three times, then total cellular RNA was extracted and viral RNA was quantified by qPCR (C and D). For the virus entry assay, these cells were then transferred to a 37°C incubator for 20 min after 1 h of incubation at 4°C and were rinsed three times with 1×PBS-HCl. Total RNA was extracted, and viral RNA was quantified by qPCR (E and F). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G and H) Wild-type, LAMTOR1 −/− (G), and LAMTOR3 −/− (H) HeLa cells were infected with EV71 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection. VP1 expression was analyzed by Western blot 12 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I) SK-N-SH cells were transfected with LAMTOR3 siRNAs and then infected with EV71 (MOI = 5) after 60 h. The knockdown efficiency of LAMTOR3 was determined by Western blotting. Viral RNA level was quantified by qPCR 5 h after infection. 3D expression was analyzed by Western blot 12 h after infection. The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (J and K) U251 cells were transfected with LAMTOR3 siRNAs and then infected with EV71-VR784 (MOI = 5; J) or EV71-695F (MOI = 5; K) after 60 h. Viral RNA was quantified by qPCR 5 h after infection. VP1 expression was analyzed by Western blot 12 h after infection. The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (L–N) RagB +/+ and RagB −/− HeLa cells were infected with EV71 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection. The knockout efficiency of RagB in HeLa cells and VP1 expression were detected by Western blot 12 h after infection (L). The quantitation analysis of Western blot was performed using ImageJ. Viral titers were determined by TCID50 assay at 12, 24, and 36 h after infection (M). Cell death induced by EV71 was detected by measuring ATP levels 18 h after infection (N). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: The Ragulator-Rag complex is required for EV71 replication. (A and B) Wild-type, LAMTOR1 −/− (A), and LAMTOR3 −/− (B) HeLa cells were infected with EV71 (MOI = 5) and the viral titers were determined by TCID50 assay at 12, 24, and 36 h after infection. (C–F) Wild-type, LAMTOR1 −/− , and LAMTOR3 −/− HeLa cells were incubated with EV71 (MOI > 100) at 4°C for 1 h. For the virus binding assay, cells were washed with 1×PBS three times, then total cellular RNA was extracted and viral RNA was quantified by qPCR (C and D). For the virus entry assay, these cells were then transferred to a 37°C incubator for 20 min after 1 h of incubation at 4°C and were rinsed three times with 1×PBS-HCl. Total RNA was extracted, and viral RNA was quantified by qPCR (E and F). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G and H) Wild-type, LAMTOR1 −/− (G), and LAMTOR3 −/− (H) HeLa cells were infected with EV71 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection. VP1 expression was analyzed by Western blot 12 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I) SK-N-SH cells were transfected with LAMTOR3 siRNAs and then infected with EV71 (MOI = 5) after 60 h. The knockdown efficiency of LAMTOR3 was determined by Western blotting. Viral RNA level was quantified by qPCR 5 h after infection. 3D expression was analyzed by Western blot 12 h after infection. The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (J and K) U251 cells were transfected with LAMTOR3 siRNAs and then infected with EV71-VR784 (MOI = 5; J) or EV71-695F (MOI = 5; K) after 60 h. Viral RNA was quantified by qPCR 5 h after infection. VP1 expression was analyzed by Western blot 12 h after infection. The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (L–N) RagB +/+ and RagB −/− HeLa cells were infected with EV71 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection. The knockout efficiency of RagB in HeLa cells and VP1 expression were detected by Western blot 12 h after infection (L). The quantitation analysis of Western blot was performed using ImageJ. Viral titers were determined by TCID50 assay at 12, 24, and 36 h after infection (M). Cell death induced by EV71 was detected by measuring ATP levels 18 h after infection (N). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Techniques: Infection, TCID50 Assay, Incubation, Virus, Binding Assay, Two Tailed Test, Expressing, Western Blot, Transfection, Knockdown, Quantitation Assay, Knock-Out

The Ragulator-Rag complex is required for EV71 replication. (A) LAMTOR3 +/+ or LAMTOR3 −/− RD cells were infected with EV71 (MOI = 2). The viral RNA was quantified by qPCR 5 h after infection and the VP1 protein was analyzed by Western blot 8 h after EV71 infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (B) WT and LAMTOR3-KO cells were infected with EV71 for 24 h. The virus titers in the culture supernatant and cells were measured by TCID50 assay. The release rate represents the proportion of virus in the supernatant relative to the total virus in WT cells and LAMTOR3-KO cells. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (C) Sequencing analysis of knockout efficiency of RagB. (D and E) RagB +/+ and RagB −/− HeLa cells were incubated with EV71 (MOI > 100) at 4°C for 1 h. For the virus binding assay, cells were washed with 1×PBS three times, then total cellular RNA was extracted and viral RNA was quantified by qPCR (D). For the virus entry assay, these cells were transferred to a 37°C incubator for 20 min after 1 h of incubation at 4°C and then rinsed three times with 1×PBS-HCl. Total RNA was extracted and viral RNA was quantified by qPCR (E). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (F–H) RagA , RagC , and RagD were silenced in HeLa cells using siRNAs and then the indicated HeLa cells were infected with EV71 (MOI = 5) 60 h after transfection. The knockdown efficiency of RagA (F), RagC (G), and RagD (H) was determined by qPCR 60 h after transfection. Viral RNA (F–H) was quantified by qPCR 5 h after infection. VP1 expression (F–H) was analyzed by Western blot 12 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: The Ragulator-Rag complex is required for EV71 replication. (A) LAMTOR3 +/+ or LAMTOR3 −/− RD cells were infected with EV71 (MOI = 2). The viral RNA was quantified by qPCR 5 h after infection and the VP1 protein was analyzed by Western blot 8 h after EV71 infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (B) WT and LAMTOR3-KO cells were infected with EV71 for 24 h. The virus titers in the culture supernatant and cells were measured by TCID50 assay. The release rate represents the proportion of virus in the supernatant relative to the total virus in WT cells and LAMTOR3-KO cells. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (C) Sequencing analysis of knockout efficiency of RagB. (D and E) RagB +/+ and RagB −/− HeLa cells were incubated with EV71 (MOI > 100) at 4°C for 1 h. For the virus binding assay, cells were washed with 1×PBS three times, then total cellular RNA was extracted and viral RNA was quantified by qPCR (D). For the virus entry assay, these cells were transferred to a 37°C incubator for 20 min after 1 h of incubation at 4°C and then rinsed three times with 1×PBS-HCl. Total RNA was extracted and viral RNA was quantified by qPCR (E). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (F–H) RagA , RagC , and RagD were silenced in HeLa cells using siRNAs and then the indicated HeLa cells were infected with EV71 (MOI = 5) 60 h after transfection. The knockdown efficiency of RagA (F), RagC (G), and RagD (H) was determined by qPCR 60 h after transfection. Viral RNA (F–H) was quantified by qPCR 5 h after infection. VP1 expression (F–H) was analyzed by Western blot 12 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Techniques: Infection, Western Blot, Two Tailed Test, Virus, TCID50 Assay, Sequencing, Knock-Out, Incubation, Binding Assay, Transfection, Knockdown, Expressing

The Ragulator-Rag complex recruits EV71 3D protein and PI4KB. (A) HeLa cells were infected with EV71 (MOI = 5) for 6 h and then the colocalization of LAMTOR3, RagB, and 3D was examined by staining cells with anti-LAMTOR3/RagB/3D antibodies followed by confocal microscopy analysis. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. (B) GST pull-down assay of in vitro translated Flag-tagged 3D and GST-tagged RagB/LAMTOR3. The quantitation analysis of Western blot was performed using ImageJ. (C) The plasmid encoding HA-3×FLAG-RagB and the plasmid encoding GFP-tagged 3D were cotransfected into 293T cells. Cell lysates were collected after 36 h for IP with anti-FLAG agarose and the interaction between 3D and RagB was detected by Western blotting. The quantitation analysis of Western blot was performed using ImageJ. (D) RD cells were harvested 6 h after EV71 infection and analyzed using the in situ PLA. Representative images of in situ PLA show the endogenous interaction between RagB and 3D (red). Nuclei were stained with DAPI. Scale bars, 10 and 40 μm. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (E) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of PI4KB and 3D was detected by staining cells with anti-PI4KB and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. The Pearson’s correlation coefficient of PI4KB and 3D was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (F) 293T cells were transfected with plasmids expressing HA-3×Flag-PI4KB, GFP-3D, and Myc-RagB, as indicated. Cell lysates were collected after 36 h and subjected to IP using anti-FLAG agarose beads. Interactions between PI4KB and 3D were detected by Western blot. The quantitation analysis of Western blot was performed using ImageJ. (G) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of LAMP1, RagB, and 3D was assessed by staining cells with anti-LAMP1/RagB/3D antibodies followed by visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. (H) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of PI4KB and LAMP1 was assessed by staining cells with anti-PI4KB and anti-LAMP1 antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. The Pearson's correlation coefficient of PI4KB and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I and J) Wild-type HeLa cells were infected with EV71 (MOI = 5). RagB −/− (I) and LAMTOR3 −/− (J) HeLa cells were infected with EV71 (MOI = 20). Co-localization of 3D with LAMP1 was assessed by staining cells with anti-LAMP1 and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. The Pearson's correlation coefficient of 3D and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: The Ragulator-Rag complex recruits EV71 3D protein and PI4KB. (A) HeLa cells were infected with EV71 (MOI = 5) for 6 h and then the colocalization of LAMTOR3, RagB, and 3D was examined by staining cells with anti-LAMTOR3/RagB/3D antibodies followed by confocal microscopy analysis. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. (B) GST pull-down assay of in vitro translated Flag-tagged 3D and GST-tagged RagB/LAMTOR3. The quantitation analysis of Western blot was performed using ImageJ. (C) The plasmid encoding HA-3×FLAG-RagB and the plasmid encoding GFP-tagged 3D were cotransfected into 293T cells. Cell lysates were collected after 36 h for IP with anti-FLAG agarose and the interaction between 3D and RagB was detected by Western blotting. The quantitation analysis of Western blot was performed using ImageJ. (D) RD cells were harvested 6 h after EV71 infection and analyzed using the in situ PLA. Representative images of in situ PLA show the endogenous interaction between RagB and 3D (red). Nuclei were stained with DAPI. Scale bars, 10 and 40 μm. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (E) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of PI4KB and 3D was detected by staining cells with anti-PI4KB and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. The Pearson’s correlation coefficient of PI4KB and 3D was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (F) 293T cells were transfected with plasmids expressing HA-3×Flag-PI4KB, GFP-3D, and Myc-RagB, as indicated. Cell lysates were collected after 36 h and subjected to IP using anti-FLAG agarose beads. Interactions between PI4KB and 3D were detected by Western blot. The quantitation analysis of Western blot was performed using ImageJ. (G) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of LAMP1, RagB, and 3D was assessed by staining cells with anti-LAMP1/RagB/3D antibodies followed by visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. (H) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of PI4KB and LAMP1 was assessed by staining cells with anti-PI4KB and anti-LAMP1 antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. The Pearson's correlation coefficient of PI4KB and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I and J) Wild-type HeLa cells were infected with EV71 (MOI = 5). RagB −/− (I) and LAMTOR3 −/− (J) HeLa cells were infected with EV71 (MOI = 20). Co-localization of 3D with LAMP1 was assessed by staining cells with anti-LAMP1 and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. The Pearson's correlation coefficient of 3D and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Techniques: Infection, Staining, Confocal Microscopy, Pull Down Assay, In Vitro, Quantitation Assay, Western Blot, Plasmid Preparation, In Situ, Two Tailed Test, Transfection, Expressing

The Ragulator-Rag complex recruits EV71 3D protein and PI4KB. (A) HeLa cells were mock infected or infected with EV71 (MOI = 5) and the changes in cellular morphology were observed under a microscope at 6 h after infection. Scale bars, 200 μm. (B) SK-N-SH cells were mock infected or infected with EV71 (MOI = 5) for 12 h. Colocalization of RagB, LAMTOR3, and 3D was detected by staining cells with anti-RagB/LAMTOR3/3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. (C–G) GST pull-down assay of in vitro translated Flag-tagged 3D and GST-tagged RagA, RagC, LAMTOR1, LAMTOR2, and LAMTOR4. The quantitation analysis of Western blot was performed using ImageJ. (H) Schematic diagram of EV71 3D protein binding to Ragulator-Rag complex. (I) SK-N-SH cells were mock-infected or infected with EV71 (MOI = 5) for 12 h. Colocalization of RagB, LAMP1, and 3D was detected by staining cells with anti-LAMP1/RagB/3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: The Ragulator-Rag complex recruits EV71 3D protein and PI4KB. (A) HeLa cells were mock infected or infected with EV71 (MOI = 5) and the changes in cellular morphology were observed under a microscope at 6 h after infection. Scale bars, 200 μm. (B) SK-N-SH cells were mock infected or infected with EV71 (MOI = 5) for 12 h. Colocalization of RagB, LAMTOR3, and 3D was detected by staining cells with anti-RagB/LAMTOR3/3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. (C–G) GST pull-down assay of in vitro translated Flag-tagged 3D and GST-tagged RagA, RagC, LAMTOR1, LAMTOR2, and LAMTOR4. The quantitation analysis of Western blot was performed using ImageJ. (H) Schematic diagram of EV71 3D protein binding to Ragulator-Rag complex. (I) SK-N-SH cells were mock-infected or infected with EV71 (MOI = 5) for 12 h. Colocalization of RagB, LAMP1, and 3D was detected by staining cells with anti-LAMP1/RagB/3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Source data are available for this figure: .

Techniques: Infection, Microscopy, Staining, Confocal Microscopy, Pull Down Assay, In Vitro, Quantitation Assay, Western Blot, Protein Binding

The lysosomal tethered Ragulator-Rag complex mediates CVA16 replication. (A–C) Wild-type, LAMTOR1 −/− -1 (A), LAMTOR3 −/− -1 (B), and RagB −/− -1 (C) HeLa cells were infected with CVA16 (MOI = 5). Viral RNA was quantified by qPCR 3 h after infection (upper panel). Expression of viral 3D was detected by Western blot analysis 12 h after infection (lower panel). The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (D–F) Wild-type, LAMTOR1 −/− -1 (D), LAMTOR3 −/− -1 (E), and RagB −/− -1 (F) HeLa cells were infected with CVA16 (MOI = 5). Cell viability was determined by measuring ATP levels 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G–I) Wild-type, LAMTOR1 −/− -1 (G), LAMTOR3 −/− -1 (H), and RagB −/− - 1(I) HeLa cells were infected with CVA16 (MOI = 5). Cleavage of GSDME and caspase3 were analyzed by Western blot 12 h after infection. (J) LAMTOR1 −/− -1+ LAMTOR1 -FLAG and LAMTOR1 −/− -1+3A-FLAG HeLa cells were infected with CVA16 (MOI = 5 and MOI = 20, respectively). Colocalization of 3D with LAMP1 was detected by staining cells with anti-LAMP1 and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm (left panel). The Pearson’s correlation coefficient of 3D and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (K and L) HeLa cells as indicated were infected with CVA16 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection (K). Cell viability was detected by measuring ATP levels 24 h after infection (L). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (M) LAMTOR3 +/+ , LAMTOR3 −/− -1, and LAMTOR3 −/− -2 HeLa cells were infected with CVA16 (MOI = 5) and viral titers were determined by TCID50 assay 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: The lysosomal tethered Ragulator-Rag complex mediates CVA16 replication. (A–C) Wild-type, LAMTOR1 −/− -1 (A), LAMTOR3 −/− -1 (B), and RagB −/− -1 (C) HeLa cells were infected with CVA16 (MOI = 5). Viral RNA was quantified by qPCR 3 h after infection (upper panel). Expression of viral 3D was detected by Western blot analysis 12 h after infection (lower panel). The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (D–F) Wild-type, LAMTOR1 −/− -1 (D), LAMTOR3 −/− -1 (E), and RagB −/− -1 (F) HeLa cells were infected with CVA16 (MOI = 5). Cell viability was determined by measuring ATP levels 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G–I) Wild-type, LAMTOR1 −/− -1 (G), LAMTOR3 −/− -1 (H), and RagB −/− - 1(I) HeLa cells were infected with CVA16 (MOI = 5). Cleavage of GSDME and caspase3 were analyzed by Western blot 12 h after infection. (J) LAMTOR1 −/− -1+ LAMTOR1 -FLAG and LAMTOR1 −/− -1+3A-FLAG HeLa cells were infected with CVA16 (MOI = 5 and MOI = 20, respectively). Colocalization of 3D with LAMP1 was detected by staining cells with anti-LAMP1 and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm (left panel). The Pearson’s correlation coefficient of 3D and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (K and L) HeLa cells as indicated were infected with CVA16 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection (K). Cell viability was detected by measuring ATP levels 24 h after infection (L). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (M) LAMTOR3 +/+ , LAMTOR3 −/− -1, and LAMTOR3 −/− -2 HeLa cells were infected with CVA16 (MOI = 5) and viral titers were determined by TCID50 assay 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: .

Techniques: Infection, Expressing, Western Blot, Quantitation Assay, Two Tailed Test, Staining, Confocal Microscopy, TCID50 Assay

The lysosomal tethered Ragulator-Rag complex mediates CVA16 replication. ( A and B) LAMTOR3 +/+ and LAMTOR3 −/− -1, LAMTOR3 −/− -2 HeLa cells were infected with HSV (MOI = 2; A) or VSV (MOI = 0.5; B) and viral titers were determined by TCID50 assay 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test.

Journal: The Journal of Cell Biology

Article Title: Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

doi: 10.1083/jcb.202303108

Figure Lengend Snippet: The lysosomal tethered Ragulator-Rag complex mediates CVA16 replication. ( A and B) LAMTOR3 +/+ and LAMTOR3 −/− -1, LAMTOR3 −/− -2 HeLa cells were infected with HSV (MOI = 2; A) or VSV (MOI = 0.5; B) and viral titers were determined by TCID50 assay 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test.

Techniques: Infection, TCID50 Assay, Two Tailed Test

Antibodies, chemicals, and plasmids used in this study.

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: Rabbit monoclonal anti‐LAMTOR3 , Cell Signaling Technology , Cat#8168; RRID:AB_10949501.

Techniques: Recombinant, In Situ, CRISPR, Control, Plasmid Preparation

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