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anti maged2 (Proteintech)

Name: anti maged2
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Proteintech anti maged2
Anti Maged2, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 91 stars, based on 6 article reviews

anti maged2 - by Bioz Stars, 2026-03

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MAGED2 depletion induces autophagy under hypoxic conditions. HEK293 cells were transfected with control or MAGED2 siRNAs. Upon confluency 24–48 h post transfection, cells were exposed to physical hypoxia overnight. Total cell lysates were separated by SDS-PAGE and blotted for p62, ATGs and LC3B detection. Blotting for HIF-1α- was carried out to confirm its induction under hypoxia. Immunocytochemistry was carried out in parallel in HEK293 cells transfected and stressed similarly prior to incubation with LC3B antibody. ( A ) Representative Western blot images demonstrate decreased p62 levels, elevated ATG5-ATG12 complex levels and a higher LC3II abundance upon MAGED2 depletion in the presence of physical hypoxia. Leupeptin assay confirmed the induced autophagy where the highest LC3II accumulation corresponded to cells where MAGED2 is knocked down. ( B ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II from the immunoblot A. All samples shown on individual blots are from the same experiment and each blot represents an example of three independent experiments. ( C ) Representative immunofluorescence images displaying LC3B staining in control and MAGED2-transfected HEK293 cells under physical hypoxia. Scale bar 5 µm. ( D ) This notion was further supported by qRT-PCR, where the quantity of ATG5 and ATG12 was determined in HEK293 cells transfected with control or MAGED2 siRNAs and exposed to hypoxia. Total mRNA was isolated, and the relative mRNA amounts of both genes were measured. Statistical significance was determined by unpaired Student’s t -test. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: MAGED2 depletion induces autophagy under hypoxic conditions. HEK293 cells were transfected with control or MAGED2 siRNAs. Upon confluency 24–48 h post transfection, cells were exposed to physical hypoxia overnight. Total cell lysates were separated by SDS-PAGE and blotted for p62, ATGs and LC3B detection. Blotting for HIF-1α- was carried out to confirm its induction under hypoxia. Immunocytochemistry was carried out in parallel in HEK293 cells transfected and stressed similarly prior to incubation with LC3B antibody. ( A ) Representative Western blot images demonstrate decreased p62 levels, elevated ATG5-ATG12 complex levels and a higher LC3II abundance upon MAGED2 depletion in the presence of physical hypoxia. Leupeptin assay confirmed the induced autophagy where the highest LC3II accumulation corresponded to cells where MAGED2 is knocked down. ( B ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II from the immunoblot A. All samples shown on individual blots are from the same experiment and each blot represents an example of three independent experiments. ( C ) Representative immunofluorescence images displaying LC3B staining in control and MAGED2-transfected HEK293 cells under physical hypoxia. Scale bar 5 µm. ( D ) This notion was further supported by qRT-PCR, where the quantity of ATG5 and ATG12 was determined in HEK293 cells transfected with control or MAGED2 siRNAs and exposed to hypoxia. Total mRNA was isolated, and the relative mRNA amounts of both genes were measured. Statistical significance was determined by unpaired Student’s t -test. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Incubation, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Isolation

Cobalt chloride induces autophagy in MAGED2-depleted HEK293 cells. HEK293 cells were transfected with control or MAGED2 siRNAs. Upon confluency 24–48 h post transfection, cells were treated with cobalt chloride (“chemical hypoxia”, CoCl 2 , 300 µM) for 14–16 h. Total cell lysates were separated by SDS-PAGE and blotted for p62, ATGs and LC3B detection. Of note, HIF-1α immunoblotting confirmed the hypoxic condition. Immunocytochemistry was carried out in parallel in HEK293 cells transfected and stressed similarly prior to incubation with LC3B antibody. ( A ) Representative Western blot images from HEK293 cells demonstrate reduced p62 levels, increased ATG5-ATG12 conjugate levels and a higher LC3II prevalence upon MAGED2 depletion. Coincubation with leupeptin (100 µM) led to an increased LC3II accumulation, which confirmed induction of autophagy. ( B ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II from immunoblot A. All samples shown on individual blots are from the same experiment, and each blot represents an example of three independent experiments. Bar graphs show mean ± SEM, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Representative immunofluorescence images displaying LC3B staining in control and MAGED2-transfected HEK293 cells treated with CoCl 2 . Scale bar 5 μM.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Cobalt chloride induces autophagy in MAGED2-depleted HEK293 cells. HEK293 cells were transfected with control or MAGED2 siRNAs. Upon confluency 24–48 h post transfection, cells were treated with cobalt chloride (“chemical hypoxia”, CoCl 2 , 300 µM) for 14–16 h. Total cell lysates were separated by SDS-PAGE and blotted for p62, ATGs and LC3B detection. Of note, HIF-1α immunoblotting confirmed the hypoxic condition. Immunocytochemistry was carried out in parallel in HEK293 cells transfected and stressed similarly prior to incubation with LC3B antibody. ( A ) Representative Western blot images from HEK293 cells demonstrate reduced p62 levels, increased ATG5-ATG12 conjugate levels and a higher LC3II prevalence upon MAGED2 depletion. Coincubation with leupeptin (100 µM) led to an increased LC3II accumulation, which confirmed induction of autophagy. ( B ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II from immunoblot A. All samples shown on individual blots are from the same experiment, and each blot represents an example of three independent experiments. Bar graphs show mean ± SEM, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Representative immunofluorescence images displaying LC3B staining in control and MAGED2-transfected HEK293 cells treated with CoCl 2 . Scale bar 5 μM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Western Blot, Immunocytochemistry, Incubation, Immunofluorescence, Staining

Autophagy is significantly induced by classical ER-stressors in MAGED2-depleted cells. Control or MAGED2 siRNAs were transfected into HEK293 cells. At 24–48 h post-transfection, cells were treated with the ER stressors 600 nM tunicamycin (Tun) overnight or 10 μM brefeldin A (BFA) for 2 h. SDS-PAGE was used to separate total cell lysates before blotting for p62, LC3B and ATGs detection. Immunocytochemistry was conducted in parallel, and HEK293 cells were stained for the accumulation of LC3B puncta after being transfected and treated with ER stressors. Representative Western blot images from HEK293 cells treated with tunicamycin ( A ) or BFA ( D ) show reduced p62 levels combined with increased levels of ATG5-ATG12 complex and higher LC3II abundance in MAGED2-depleted cells. Leupeptin treatment led to the highest LC3II accumulation because of blocked autophagic flux. ( B , E ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II of the immunoblots ( A , D ) respectively. All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C , F ) LC3B staining in control and MAGED2-transfected HEK 293 cells treated with tunicamycin or BFA, respectively. The scale bar is 5 μM.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Autophagy is significantly induced by classical ER-stressors in MAGED2-depleted cells. Control or MAGED2 siRNAs were transfected into HEK293 cells. At 24–48 h post-transfection, cells were treated with the ER stressors 600 nM tunicamycin (Tun) overnight or 10 μM brefeldin A (BFA) for 2 h. SDS-PAGE was used to separate total cell lysates before blotting for p62, LC3B and ATGs detection. Immunocytochemistry was conducted in parallel, and HEK293 cells were stained for the accumulation of LC3B puncta after being transfected and treated with ER stressors. Representative Western blot images from HEK293 cells treated with tunicamycin ( A ) or BFA ( D ) show reduced p62 levels combined with increased levels of ATG5-ATG12 complex and higher LC3II abundance in MAGED2-depleted cells. Leupeptin treatment led to the highest LC3II accumulation because of blocked autophagic flux. ( B , E ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II of the immunoblots ( A , D ) respectively. All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C , F ) LC3B staining in control and MAGED2-transfected HEK 293 cells treated with tunicamycin or BFA, respectively. The scale bar is 5 μM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Staining, Western Blot

Genotoxic stress enhances autophagy in MAGED2-depleted cells. HEK293 cells were transfected with control or MAGED2 siRNAs. Cells were treated 24–48 h post-transfection with 10 µM camptothecin (CPT) overnight. Total cell proteins were separated using SDS-PAGE and then immunoblotted for p62, ATGs and LC3B detection. In parallel, immunocytochemistry for HEK293 cells was carried out to stain for LC3B puncta following the same protocol. ( A ) Representative Western blot images from HEK293 cells treated with CPT show decreased p62 expression, increased levels of ATG5-ATG12 complex and higher LC3II abundance upon MAGED2 depletion. Treatment with leupeptin blocked the autophagic flux and resulted in the highest LC3II accumulation when MAGED2 was depleted. ( B ) Densitometric analysis of p62, ATG5-ATG12 complex and LC3II from the immunoblots in ( A ). All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Representative immunofluorescence images showing LC3B staining in control and MAGED2-transfected HEK293 cells upon CPT treatment. Scale bar 5 µM.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Genotoxic stress enhances autophagy in MAGED2-depleted cells. HEK293 cells were transfected with control or MAGED2 siRNAs. Cells were treated 24–48 h post-transfection with 10 µM camptothecin (CPT) overnight. Total cell proteins were separated using SDS-PAGE and then immunoblotted for p62, ATGs and LC3B detection. In parallel, immunocytochemistry for HEK293 cells was carried out to stain for LC3B puncta following the same protocol. ( A ) Representative Western blot images from HEK293 cells treated with CPT show decreased p62 expression, increased levels of ATG5-ATG12 complex and higher LC3II abundance upon MAGED2 depletion. Treatment with leupeptin blocked the autophagic flux and resulted in the highest LC3II accumulation when MAGED2 was depleted. ( B ) Densitometric analysis of p62, ATG5-ATG12 complex and LC3II from the immunoblots in ( A ). All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Representative immunofluorescence images showing LC3B staining in control and MAGED2-transfected HEK293 cells upon CPT treatment. Scale bar 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Staining, Western Blot, Expressing, Immunofluorescence

Nutritional stress promotes autophagy in MAGED2-depleted cells. Control or MAGED2 siRNAs were transfected into HEK293 cells. Cells were treated 24–48 h post transfection with 4 mM 2-Deoxy-D-glucose (2DG) for 30 min. SDS-PAGE was used to separate total cell lysates, which were next blotted for p62, ATGs and LC3B detection. Immunocytochemistry was conducted as mentioned before, and both HeLa and HEK293 cells were stained for the accumulation of LC3B puncta. ( A ) Representative Western blot images from HEK293 cells treated with 2DG shows decreased p62 levels, elevated expression of ATG5-ATG12 complex, higher LC3II abundance upon MAGED2 depletion and the highest ratio when co-incubating with Leupeptin. ( B ) Densitometric analysis of P62, ATGs and LC3II from the immunoblots in ( A ). All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01. ( C ) Representative immunofluorescence images showing LC3B staining in control and MAGED2-transfected HEK293 cells upon 2DG treatment. Scale bar 5 µM.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Nutritional stress promotes autophagy in MAGED2-depleted cells. Control or MAGED2 siRNAs were transfected into HEK293 cells. Cells were treated 24–48 h post transfection with 4 mM 2-Deoxy-D-glucose (2DG) for 30 min. SDS-PAGE was used to separate total cell lysates, which were next blotted for p62, ATGs and LC3B detection. Immunocytochemistry was conducted as mentioned before, and both HeLa and HEK293 cells were stained for the accumulation of LC3B puncta. ( A ) Representative Western blot images from HEK293 cells treated with 2DG shows decreased p62 levels, elevated expression of ATG5-ATG12 complex, higher LC3II abundance upon MAGED2 depletion and the highest ratio when co-incubating with Leupeptin. ( B ) Densitometric analysis of P62, ATGs and LC3II from the immunoblots in ( A ). All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01. ( C ) Representative immunofluorescence images showing LC3B staining in control and MAGED2-transfected HEK293 cells upon 2DG treatment. Scale bar 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Staining, Western Blot, Expressing, Immunofluorescence

GNAS depletion induces autophagy under hypoxic conditions. Control or GNAS siRNAs were transfected into HEK293 cells. Upon confluency, 24–48 h post transfection, hypoxic stress was applied overnight for one set in a modular chamber while the other set was kept in normoxic conditions. Cells were then lysed and blotted for detection of p62, LC3B and autophagy-related genes. HIF-1α immunoblot confirmed hypoxia. ( A ) A representative Western blot from HEK293 cells demonstrates that GNAS depletion induced autophagy significantly under hypoxia where the low p62 levels and ATGs upregulation were accompanied by a higher conversion to the lipidated form LC3II under hypoxia. ( B ) Densitometric analysis of p62, ATG5-ATG12 complex and the LC3II from the immunoblot A. All samples shown on individual blots are from the same experiment, and each blot represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Immunocytochemistry for HEK293 cells transfected with either control, MAGED2 or GNAS siRNAs and exposed to physical hypoxia for 14–16 h show the accumulation of LC3B puncta. Similar to MAGED2-depletion, knockdown of GNAS also led to an increased abundance of LC3B positive puncta. Scale bar is 5 µM.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: GNAS depletion induces autophagy under hypoxic conditions. Control or GNAS siRNAs were transfected into HEK293 cells. Upon confluency, 24–48 h post transfection, hypoxic stress was applied overnight for one set in a modular chamber while the other set was kept in normoxic conditions. Cells were then lysed and blotted for detection of p62, LC3B and autophagy-related genes. HIF-1α immunoblot confirmed hypoxia. ( A ) A representative Western blot from HEK293 cells demonstrates that GNAS depletion induced autophagy significantly under hypoxia where the low p62 levels and ATGs upregulation were accompanied by a higher conversion to the lipidated form LC3II under hypoxia. ( B ) Densitometric analysis of p62, ATG5-ATG12 complex and the LC3II from the immunoblot A. All samples shown on individual blots are from the same experiment, and each blot represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Immunocytochemistry for HEK293 cells transfected with either control, MAGED2 or GNAS siRNAs and exposed to physical hypoxia for 14–16 h show the accumulation of LC3B puncta. Similar to MAGED2-depletion, knockdown of GNAS also led to an increased abundance of LC3B positive puncta. Scale bar is 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, Western Blot, Immunocytochemistry

Forskolin reverses the induction of autophagy under stress conditions upon MAGED2 depletion. HeLa and HEK293 cells were both transfected with control and MAGED2 siRNA. After 24 to 48 h upon confluency, the media was changed to DMEM as control or DMEM containing 10 µM FSK before subjecting all cells to physical hypoxia overnight. SDS-PAGE was used to separate total cell proteins, which were further immunoblotted for LC3B detection. Hypoxic condition was verified by blotting for HIF-1α. Moreover, immunocytochemistry for HEK293 cells transfected with either control or MAGED2 siRNA and exposed upon confluency to overnight physical hypoxia with or without the addition of 10 µM of FSK was performed and the accumulation of LC3B puncta was analyzed. ( A , B ) Representative Western blot images from ( A ) Hela and ( B ) HEK293 cells treated with 10 µM FSK show a reduction in LC3B expression and a diminished autophagy induction upon FSK treatment. The promoted autophagy seen when MAGED2 is knocked-down is rendered to control levels by FSK addition. ( C , D ) Densitometric analysis of LC3B immunoblots in ( A , B ), respectively. All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( E ) Immunocytochemistry confirms that FSK addition to HEK293 cells prior to hypoxic stress reversed the observed autophagic induction, and the accumulation of puncta was rendered to control levels. Scale bar is 5 µM.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Forskolin reverses the induction of autophagy under stress conditions upon MAGED2 depletion. HeLa and HEK293 cells were both transfected with control and MAGED2 siRNA. After 24 to 48 h upon confluency, the media was changed to DMEM as control or DMEM containing 10 µM FSK before subjecting all cells to physical hypoxia overnight. SDS-PAGE was used to separate total cell proteins, which were further immunoblotted for LC3B detection. Hypoxic condition was verified by blotting for HIF-1α. Moreover, immunocytochemistry for HEK293 cells transfected with either control or MAGED2 siRNA and exposed upon confluency to overnight physical hypoxia with or without the addition of 10 µM of FSK was performed and the accumulation of LC3B puncta was analyzed. ( A , B ) Representative Western blot images from ( A ) Hela and ( B ) HEK293 cells treated with 10 µM FSK show a reduction in LC3B expression and a diminished autophagy induction upon FSK treatment. The promoted autophagy seen when MAGED2 is knocked-down is rendered to control levels by FSK addition. ( C , D ) Densitometric analysis of LC3B immunoblots in ( A , B ), respectively. All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( E ) Immunocytochemistry confirms that FSK addition to HEK293 cells prior to hypoxic stress reversed the observed autophagic induction, and the accumulation of puncta was rendered to control levels. Scale bar is 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Western Blot, Expressing

Proposed model for the role of MAGED2 under stress conditions (created with BioRender.com). Under stress, MAGED2 inhibits MDM2-dependent ubiquitination and endocytosis of Gαs. This ensures activation of the adenylate cyclase and cAMP generation and activation of PKA under stress. Reduced PKA activity impairs regulation of autophagy mediated by the cAMP/PKA pathway.

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Proposed model for the role of MAGED2 under stress conditions (created with BioRender.com). Under stress, MAGED2 inhibits MDM2-dependent ubiquitination and endocytosis of Gαs. This ensures activation of the adenylate cyclase and cAMP generation and activation of PKA under stress. Reduced PKA activity impairs regulation of autophagy mediated by the cAMP/PKA pathway.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Activation Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, Lysis, SYBR Green Assay, Real-time Polymerase Chain Reaction, Software

Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet:

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Recombinant, Magnetic Beads, Protease Inhibitor, Cotransfection, Expressing, Transfection, Labeling, Software

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Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet: Novel Gαs AHD-binding protein that facilitates AHD closing (A) Representative confocal images of HEK293T cells transiently expressing Gαs AHD-FLAG. HEK293T cells were stained with antibodies against FLAG (red) and MAGE D2 (green). Blue represents Hoechst 33342. Right: Representative co-localization tracer profile along the line indicated in the inset. Scale bar, 10 μm. (B) Immunoblot (IB) analysis of FLAG immunoprecipitates (IP line) and cell lysates (Input line) from HEK293T cells transiently co-expressing Gαs AHD-FLAG and MAGE D2-turboGFP. (C) Binding curve of the Gαs AHD with the MAGE D2 MHD analyzed by microscale thermophoresis (MST). A titration series of the Gαs AHD was performed, whereas the concentration of fluorescence-labeled MAGE D2 MHD was fixed (see for details). Error bars represent the standard error of the mean of more than three independent experiments. (D) Time-resolved tryptophan-induced bimane quenching analysis of the β 2 AR–Gs complex with or without the MAGE D2 MHD after GTPγS addition. The data are mean of the normalized fluorescence value of three independently labeled experiments and error bars represent the standard error of the mean. See also Figures S3 , , and .

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Binding Assay, Expressing, Staining, Western Blot, Microscale Thermophoresis, Titration, Concentration Assay, Fluorescence, Labeling

Summary cartoon illustrating the proposed sequence of events after GTP binding to a GPCR–Gs complex with or without MAGE D2 An agonist-activated receptor (R) induces GDP release from Gs, and GTP quickly binds to the empty nucleotide-binding pocket (step i). Upon binding of GTP, Gαs rapidly dissociates from the receptor and Gβγ (step ii). The AHD adopts a long-lived intermediate state until it is fully closed, a process that occurs slowly (step iii-a). Closing of the AHD is accelerated in the presence of MAGE D2 (step iii-b). RD indicates the Ras-like GTPase domain, and AHD indicates the α-helical domain.

Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet: Summary cartoon illustrating the proposed sequence of events after GTP binding to a GPCR–Gs complex with or without MAGE D2 An agonist-activated receptor (R) induces GDP release from Gs, and GTP quickly binds to the empty nucleotide-binding pocket (step i). Upon binding of GTP, Gαs rapidly dissociates from the receptor and Gβγ (step ii). The AHD adopts a long-lived intermediate state until it is fully closed, a process that occurs slowly (step iii-a). Closing of the AHD is accelerated in the presence of MAGE D2 (step iii-b). RD indicates the Ras-like GTPase domain, and AHD indicates the α-helical domain.

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Sequencing, Binding Assay

Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet:

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Recombinant, Magnetic Beads, Protease Inhibitor, Cotransfection, Expressing, Transfection, Labeling, Software

SARS-CoV-2 Mpro cleaves MAGED2 at Gln-263. ( A ) Putative Mpro cleavage sites in SARS-CoV-2 non-structural proteins and MAGED2. ( B and C ) HEK293T cells were co-transfected with human MAGED2 or Q263N mutant with Flag tag at C-terminal and HA-tagged SARS-CoV-2 Mpro or a proteolytically inactive mutant Mpro (C145A). Lysates from transfected cells were prepared for immunoblotting with antibodies, as indicated. ( D ) MAGED2 mutant with Flag tag at C-terminal and HA-tagged Mpro were co-expressed in HEK293T cells. Lysates from transfected cells were prepared for immunoblotting with antibodies, as indicated. ( E ) MAGED2 cleavage assay in vitro . Purified MAGED2 and Mpro wild-type (WT) or C145A mutant proteins were incubated in vitro and analyzed by Coomassie blue staining. One star is MAGED2 N , and two stars indicate MAGED2 C . ( F ) A549-hACE2 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 or 1, and immunoblot was performed at 24-h post-infection. Red star indicates cleaved MAGED2. Each experiment was independently repeated three times with similar results, and the representative images are shown.

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: SARS-CoV-2 Mpro cleaves MAGED2 at Gln-263. ( A ) Putative Mpro cleavage sites in SARS-CoV-2 non-structural proteins and MAGED2. ( B and C ) HEK293T cells were co-transfected with human MAGED2 or Q263N mutant with Flag tag at C-terminal and HA-tagged SARS-CoV-2 Mpro or a proteolytically inactive mutant Mpro (C145A). Lysates from transfected cells were prepared for immunoblotting with antibodies, as indicated. ( D ) MAGED2 mutant with Flag tag at C-terminal and HA-tagged Mpro were co-expressed in HEK293T cells. Lysates from transfected cells were prepared for immunoblotting with antibodies, as indicated. ( E ) MAGED2 cleavage assay in vitro . Purified MAGED2 and Mpro wild-type (WT) or C145A mutant proteins were incubated in vitro and analyzed by Coomassie blue staining. One star is MAGED2 N , and two stars indicate MAGED2 C . ( F ) A549-hACE2 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 or 1, and immunoblot was performed at 24-h post-infection. Red star indicates cleaved MAGED2. Each experiment was independently repeated three times with similar results, and the representative images are shown.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Transfection, Mutagenesis, FLAG-tag, Western Blot, Cleavage Assay, In Vitro, Purification, Incubation, Staining, Infection

MAGED2 cleavage by Mpro is conserved in multiple mammalian species and coronaviruses. ( A ) A phylogenetic tree was constructed based on the protein sequences of MAGED2 orthologs by using the neighbor-joining method conducted in program MEGA6. MAGED2 residues neighboring Mpro cleavage site from human, rhesus macaque, White-tufted-ear marmoset, tufted capuchin, Sunda flying lemur, Brandt’s bat, goat, cattle, horse, Malayan pangolin, dog, domestic ferret, domestic cat, and house mouse are aligned. ( B to E ) HEK293T cells were transfected with MAGED2 orthologs as indicated species. The uncleaved or cleaved protein band intensity was quantitatively analyzed using ImageJ. Cleavage efficiency = cleaved products/(cleaved products + uncleaved protein) × 100% ( B ), MAGED2 mutant S264P ( C ), and HA-tagged Mpro from SARS-CoV-2, other coronavirus (SARS-CoV or MERS-CoV) ( D ), or SARS-CoV-2 variants (Beta or Omicron) ( E ). Lysates of transfected cells were analyzed by immunoblotting with the antibodies indicated on the left. Western blots are quantified with ImageJ. Each experiment was independently repeated three times with similar results, and the representative images are shown. Values are means plus standard deviations (error bars) from one representative experiment with three biological replicate samples. **, P < 0.01 by one-way analysis of variance.

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 cleavage by Mpro is conserved in multiple mammalian species and coronaviruses. ( A ) A phylogenetic tree was constructed based on the protein sequences of MAGED2 orthologs by using the neighbor-joining method conducted in program MEGA6. MAGED2 residues neighboring Mpro cleavage site from human, rhesus macaque, White-tufted-ear marmoset, tufted capuchin, Sunda flying lemur, Brandt’s bat, goat, cattle, horse, Malayan pangolin, dog, domestic ferret, domestic cat, and house mouse are aligned. ( B to E ) HEK293T cells were transfected with MAGED2 orthologs as indicated species. The uncleaved or cleaved protein band intensity was quantitatively analyzed using ImageJ. Cleavage efficiency = cleaved products/(cleaved products + uncleaved protein) × 100% ( B ), MAGED2 mutant S264P ( C ), and HA-tagged Mpro from SARS-CoV-2, other coronavirus (SARS-CoV or MERS-CoV) ( D ), or SARS-CoV-2 variants (Beta or Omicron) ( E ). Lysates of transfected cells were analyzed by immunoblotting with the antibodies indicated on the left. Western blots are quantified with ImageJ. Each experiment was independently repeated three times with similar results, and the representative images are shown. Values are means plus standard deviations (error bars) from one representative experiment with three biological replicate samples. **, P < 0.01 by one-way analysis of variance.

Techniques: Construct, Transfection, Mutagenesis, Western Blot

MAGED2 is a restriction factor that inhibits SARS-CoV-2 infection. ( A ) Caco-2-N cells were transduced with sgRNA targeting MAGED2. Whole-cell lysate was analyzed by immunoblotting assay at 5-day post-transduction. ( B and C ) WT or MAGED2-depleted Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. After 24 h, cells were analyzed by flow cytometry to determine the percentage of SARS-CoV-2 GFP/ΔN trVLP-infected cells. Data are normalized with non-targeting control ( B ). Meanwhile, intracellular RNAs were purified for RT-qPCR assay to quantify SARS-CoV-2 genomic RNAs ( C ). ( D ) Human MAGED2 was ectopically expressed in Caco-2-N cells by lentiviral transduction, and the cells were subsequently infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. Cells were analyzed by flow cytometry at 24-h post-infection to determine the percentage of the trVLP-infected cells. ( E and F ) MAGED2 knockout Caco-2 cells were infected with SARS-CoV-2 authentic virus at an MOI of 0.1. After 24 h, viral particles in the supernatant were titrated ( E ), and intracellular RNAs were purified for RT-qPCR assay to quantify SARS-CoV-2 subgenomic E RNAs ( F ). Values are means + standard deviations (error bars) from one representative experiment with three biological replicate samples, and each experiment was repeated three times. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance.

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 is a restriction factor that inhibits SARS-CoV-2 infection. ( A ) Caco-2-N cells were transduced with sgRNA targeting MAGED2. Whole-cell lysate was analyzed by immunoblotting assay at 5-day post-transduction. ( B and C ) WT or MAGED2-depleted Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. After 24 h, cells were analyzed by flow cytometry to determine the percentage of SARS-CoV-2 GFP/ΔN trVLP-infected cells. Data are normalized with non-targeting control ( B ). Meanwhile, intracellular RNAs were purified for RT-qPCR assay to quantify SARS-CoV-2 genomic RNAs ( C ). ( D ) Human MAGED2 was ectopically expressed in Caco-2-N cells by lentiviral transduction, and the cells were subsequently infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. Cells were analyzed by flow cytometry at 24-h post-infection to determine the percentage of the trVLP-infected cells. ( E and F ) MAGED2 knockout Caco-2 cells were infected with SARS-CoV-2 authentic virus at an MOI of 0.1. After 24 h, viral particles in the supernatant were titrated ( E ), and intracellular RNAs were purified for RT-qPCR assay to quantify SARS-CoV-2 subgenomic E RNAs ( F ). Values are means + standard deviations (error bars) from one representative experiment with three biological replicate samples, and each experiment was repeated three times. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance.

Techniques: Infection, Transduction, Western Blot, Flow Cytometry, Purification, Quantitative RT-PCR, Knock-Out, Virus

MAGED2 inhibits SARS-CoV-2 genome replication but not restricts viral entry, assembly, and release. ( A and B ) HeLa-ACE2 cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2, ACE2, and β-actin antibodies. The HeLa-ACE2 cells with or without MAGED2 depletion were infected with MLV retroviral particles (Fluc as the reporter) pseduotyped with SARS-CoV-2 spike (MLV SARS-CoV-2pp). Fluc activity was measured at 48-h post-infection. 1F11 is SARS-CoV-2 neutralizing antibody as the positive control. ( C ) Schematic representation of SARS-CoV-2 Gluc replicon RNA genome. ( D and E ) Caco-2-N cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-Actin antibodies. Then, the cells with or without MAGED2 genetic ablation were transfected with SARS-CoV-2 Gluc WT or SAA (RdRp inactive mutant) replicon RNAs, and Gluc activity was assayed at 48-h post-transfection. ( F ) Schematic representation of VLP production and detection. ( G and H ) HEK293T cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-actin antibodies. Then, the HEK293T cells with or without MAGED2 knockout in 10 cm dish were transfected with equal amounts of plasmids (24 µg in total) encoding the SARS-CoV-2 S, E, M, and HiBiT-N proteins. After 24 h, cell culture supernatants were collected. VLPs separated by 10%–60% sucrose gradient centrifugation were measured with Nano-Glo luciferase kit. All data are representative of three independent experiments. Values are means + standard deviations (error bars) ( n = 3). *, P < 0.05; **, P < 0.01; n.s., not significantly different by one-way analysis of variance.

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 inhibits SARS-CoV-2 genome replication but not restricts viral entry, assembly, and release. ( A and B ) HeLa-ACE2 cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2, ACE2, and β-actin antibodies. The HeLa-ACE2 cells with or without MAGED2 depletion were infected with MLV retroviral particles (Fluc as the reporter) pseduotyped with SARS-CoV-2 spike (MLV SARS-CoV-2pp). Fluc activity was measured at 48-h post-infection. 1F11 is SARS-CoV-2 neutralizing antibody as the positive control. ( C ) Schematic representation of SARS-CoV-2 Gluc replicon RNA genome. ( D and E ) Caco-2-N cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-Actin antibodies. Then, the cells with or without MAGED2 genetic ablation were transfected with SARS-CoV-2 Gluc WT or SAA (RdRp inactive mutant) replicon RNAs, and Gluc activity was assayed at 48-h post-transfection. ( F ) Schematic representation of VLP production and detection. ( G and H ) HEK293T cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-actin antibodies. Then, the HEK293T cells with or without MAGED2 knockout in 10 cm dish were transfected with equal amounts of plasmids (24 µg in total) encoding the SARS-CoV-2 S, E, M, and HiBiT-N proteins. After 24 h, cell culture supernatants were collected. VLPs separated by 10%–60% sucrose gradient centrifugation were measured with Nano-Glo luciferase kit. All data are representative of three independent experiments. Values are means + standard deviations (error bars) ( n = 3). *, P < 0.05; **, P < 0.01; n.s., not significantly different by one-way analysis of variance.

Techniques: Transduction, Western Blot, Infection, Activity Assay, Positive Control, Transfection, Mutagenesis, Knock-Out, Cell Culture, Gradient Centrifugation, Luciferase

MAGED2 interacts with viral N protein to disturb the association of N with viral RNA genome. ( A ) Caco-2 cells were infected with SARS-CoV-2 authentic virus at an MOI of 0.1. After 24 h, cells were collected and lysed. Cell lysates were immunoprecipitated with MAGED2 antibody or IgG control with/without 50 µg/mL RNase A treatment. Immunoprecipitants were subjected for immunoblotting assay with MAGED2 and N antibodies. ( B ) HA-N protein and Flag tagged MAGED2, MAGED2 N (1-263 aa)-EGFP or MAGED2 C (264-606 aa) were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated by Flag antibody-conjugated magnetic beads. Immunoprecipitants were subjected for immunoblotting assay with Flag and HA antibodies. ( C and D ) Schematic representation of RNA immunoprecipitation (RIP) assay. SARS-CoV-2 Gluc replicon RNAs, plasmids encoding GFP-Flag or N-Flag and HA-MAGED2 full-length or C-terminal truncation were co-electroporated into HEK293T cells. RIP was performed at 24-h post-electroporation as indicated, and RT-qPCR assay was conducted to determine the RNA abundances. The precipitated RNA was normalized with input. ( E ) Subcellular localization of MAGED2 full-length and its truncations. Flag-tagged MAGED2 full-length, MAGED2 N , MAGED2 C , or MAGED2 N (ΔNLS) was expressed in Caco-2 cells by lentiviral transduction. Cells were stained with Flag antibody, and the nuclei were stained with DAPI. ( F ) HA-tagged N protein and Flag-tagged MAGED2 N (1-263 aa) or MAGED2 N (ΔNLS) were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated by Flag antibody-conjugated magnetic beads. Immunoprecipitants were subjected for immunoblotting assay with Flag and HA antibodies. ( G ) Human MAGED2 full-length or its truncations were ectopically expressed in Caco-2-N cells by lentiviral transduction, and the cells were subsequently infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. Cells were analyzed by flow cytometry at 24-h post-infection to determine the percentage of the trVLP-infected cells. Values are means + standard deviations (error bars) ( n = 3). ***, P < 0.001; n.s., not significantly different by one-way analysis of variance.

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 interacts with viral N protein to disturb the association of N with viral RNA genome. ( A ) Caco-2 cells were infected with SARS-CoV-2 authentic virus at an MOI of 0.1. After 24 h, cells were collected and lysed. Cell lysates were immunoprecipitated with MAGED2 antibody or IgG control with/without 50 µg/mL RNase A treatment. Immunoprecipitants were subjected for immunoblotting assay with MAGED2 and N antibodies. ( B ) HA-N protein and Flag tagged MAGED2, MAGED2 N (1-263 aa)-EGFP or MAGED2 C (264-606 aa) were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated by Flag antibody-conjugated magnetic beads. Immunoprecipitants were subjected for immunoblotting assay with Flag and HA antibodies. ( C and D ) Schematic representation of RNA immunoprecipitation (RIP) assay. SARS-CoV-2 Gluc replicon RNAs, plasmids encoding GFP-Flag or N-Flag and HA-MAGED2 full-length or C-terminal truncation were co-electroporated into HEK293T cells. RIP was performed at 24-h post-electroporation as indicated, and RT-qPCR assay was conducted to determine the RNA abundances. The precipitated RNA was normalized with input. ( E ) Subcellular localization of MAGED2 full-length and its truncations. Flag-tagged MAGED2 full-length, MAGED2 N , MAGED2 C , or MAGED2 N (ΔNLS) was expressed in Caco-2 cells by lentiviral transduction. Cells were stained with Flag antibody, and the nuclei were stained with DAPI. ( F ) HA-tagged N protein and Flag-tagged MAGED2 N (1-263 aa) or MAGED2 N (ΔNLS) were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated by Flag antibody-conjugated magnetic beads. Immunoprecipitants were subjected for immunoblotting assay with Flag and HA antibodies. ( G ) Human MAGED2 full-length or its truncations were ectopically expressed in Caco-2-N cells by lentiviral transduction, and the cells were subsequently infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. Cells were analyzed by flow cytometry at 24-h post-infection to determine the percentage of the trVLP-infected cells. Values are means + standard deviations (error bars) ( n = 3). ***, P < 0.001; n.s., not significantly different by one-way analysis of variance.

Techniques: Infection, Virus, Immunoprecipitation, Western Blot, Transfection, Magnetic Beads, Electroporation, Quantitative RT-PCR, Transduction, Staining, Flow Cytometry

Mpro cleaves MAGED2 to antagonize its antiviral activity. Model depicts that MAGED2 restricts SARS-CoV-2 replication by decreasing the interaction between N protein and viral genome through its N-terminal region. Mpro cleaves MAGED2 at Gln-263, and MAGED2 N translocated into the nucleus, which relieving its antiviral effect.

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: Mpro cleaves MAGED2 to antagonize its antiviral activity. Model depicts that MAGED2 restricts SARS-CoV-2 replication by decreasing the interaction between N protein and viral genome through its N-terminal region. Mpro cleaves MAGED2 at Gln-263, and MAGED2 N translocated into the nucleus, which relieving its antiviral effect.

Techniques: Activity Assay

Journal: Cells

Article Title: Reciprocal Regulation of MAGED2 and HIF-1α Augments Their Expression under Hypoxia: Role of cAMP and PKA Type II

doi: 10.3390/cells11213424

Figure Lengend Snippet: Reagents and tools.

Article Snippet: MAGED2, Human melanoma antigen family D, 2, Real-Time PCR Primer Set , Biomol , VHPS-5486.

Techniques: Recombinant, Lysis, cDNA Synthesis, SYBR Green Assay, Control, Real-time Polymerase Chain Reaction, Software

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