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flag becn1 24388 plasmids (Addgene inc)

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Addgene inc flag becn1 24388 plasmids
Flag Becn1 24388 Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna4+becn1+flag/pcDNA4-Beclin1+(FL)+(Plasmid+%2324388)/pmc12599409-52-3-9
Average 93 stars, based on 25 article reviews

flag becn1 24388 plasmids - by Bioz Stars, 2026-10

93/100 stars

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Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function.
Article Snippet: The NP40 detergent solution (85124) was purchased from Thermo, and protein A beads (17-0963-02) were from GE Health Care. .. Plasmids The GFP-ZFYVE1 constructs were kindly provided by Li Yu (Tsinghua University). pcDNA4-BECN1-Flag (24388) and pcDNA4-PIK3C3-Flag (24398) were purchased from Addgene. pEF-ATG14 and pEF-UVRAG were constructed in the pEF6BSD-myc/his-B plasmid (Invitrogen, V962-20). ..

Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor
Article Snippet: .. The following DNA or siRNA/shRNA constructs were also used in this work: empty pEGFP (Clontech), pEGFP-α-synuclein A53T and pcDNA3.1-myc-6XHis (Invitrogen); pCMV6-XL5-MCCC1 (#SC113201) from Origene; pcDNA3.1-EP300-6XHis (#23252), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA4-BECN1-Flag (#24388) from Addgene. .. Huntington’s disease gene exon 1 fragment with 74 poly-Q repeats in pEGFP-C1 (Clontech; EGFP-HTT(Q74)) was previously characterized .

Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
Article Snippet: The NP40 detergent solution (85124) was purchased from Thermo, and protein A beads (17-0963-02) were from GE Health Care. .. The GFP-ZFYVE1 constructs were kindly provided by Li Yu (Tsinghua University). pcDNA4-BECN1-Flag (24388) and pcDNA4-PIK3C3-Flag (24398) were purchased from Addgene. pEF-ATG14 and pEF-UVRAG were constructed in the pEF6-BSD-myc/his-B plasmid (Invitrogen, V962-20). ..

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes
Article Snippet: .. The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene. ..

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner
Article Snippet: .. The following constructs were obtained through Addgene: pEMD-C1-GFP-BCL2 fusion genes (GFP-BCL2, 17999; GFP-BCL2-Cb5, 18000; and GFP-BCL2-Maob, 18001; all obtained from Dr. Clark Distelhorst’s lab), pRK5-PRKN-MYC (7612, Dr. David Root’s lab), pC3-EGFP-LC3B (11546, Dr. Karla Kirkegaard’s lab), pMX-GFP-ZFYVE1 (38269, Dr. Noboru Mizushima’s lab), and pcDNA4-BECN1-Flag (24388, Dr. Qing Zhong’s lab). .. The protein GST was HA-tagged and cloned into pcDNA5 (ThermoFisher Scientific, V601020), as previously described [ ].

Plasmid Preparation:

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KIT interacts with BECN1, altering its interactome and inhibiting PIK3C3 kinase activity. (A and B) Representative immunoblots showing the interaction between BECN1 and KIT in MCPyV + and MCPyV − cells co-immunoprecipitated with anti-KIT (A) or anti-BECN1 (B). IP, immunoprecipitation. IB, immunoblotting. (C) Schematic illustration of the BECN1 protein with its annotated functional domains and different BECN1 expression constructs. The results of the KIT interaction were based on the experiments described in (D). (D) Representative immunoblots showing the interaction between BECN1 and KIT in KIT-HEK293 cells expressing different flag-tagged BECN1 expression constructs. IgG IP was used as a negative control. Quantification of the KIT pulled down by FLAG IP is shown on the right panel ( n = 5). (E) Immunoblots showing the effect of LT339 and LT339 W209A on BECN1 interactome. The IP efficiency is shown in figure S3C. The enrichment of BCL2 and PIK3C3 in FLAG IP is shown below the IB ( n = 8). BCL2 and PIK3C3 in FLAG IP was quantified by normalization to their respective co-IP FLAG level and compared to LT339. (F) Immunoblot analysis of BECN1 interactomes in KIT-HEK293 cells co-transfected with FLAG-tagged three-tyrosine phosphomimetic (EEE) or non-phosphorylatable (FFF) BECN1 expression constructs together with LT339 or LT339 W209A . Quantifications of the MAP1LC3B-II and SQSTM1 are shown below the corresponding IB. (G) Representative images showing the effect of LT339 and LT339 W209A on GFP-FYVE dots (green). Quantification of the GFP-FYVE dots were counted from three independent experiments (CTR: n = 68; LT339: n = 38; LT339 W209A : n = 39). Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (H) The PIK3C3 kinase activity was evaluated in the FLAG IP lysates from cells expressing LT339 or LT339 W209A using the PI3K lipid kinase assay ( n = 4). (D, E, G and H) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant, one-way ANOVA with post-hoc Dunnett’s (D) or Tukey’s (G and H), and paired t -test (E).

Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: KIT interacts with BECN1, altering its interactome and inhibiting PIK3C3 kinase activity. (A and B) Representative immunoblots showing the interaction between BECN1 and KIT in MCPyV + and MCPyV − cells co-immunoprecipitated with anti-KIT (A) or anti-BECN1 (B). IP, immunoprecipitation. IB, immunoblotting. (C) Schematic illustration of the BECN1 protein with its annotated functional domains and different BECN1 expression constructs. The results of the KIT interaction were based on the experiments described in (D). (D) Representative immunoblots showing the interaction between BECN1 and KIT in KIT-HEK293 cells expressing different flag-tagged BECN1 expression constructs. IgG IP was used as a negative control. Quantification of the KIT pulled down by FLAG IP is shown on the right panel ( n = 5). (E) Immunoblots showing the effect of LT339 and LT339 W209A on BECN1 interactome. The IP efficiency is shown in figure S3C. The enrichment of BCL2 and PIK3C3 in FLAG IP is shown below the IB ( n = 8). BCL2 and PIK3C3 in FLAG IP was quantified by normalization to their respective co-IP FLAG level and compared to LT339. (F) Immunoblot analysis of BECN1 interactomes in KIT-HEK293 cells co-transfected with FLAG-tagged three-tyrosine phosphomimetic (EEE) or non-phosphorylatable (FFF) BECN1 expression constructs together with LT339 or LT339 W209A . Quantifications of the MAP1LC3B-II and SQSTM1 are shown below the corresponding IB. (G) Representative images showing the effect of LT339 and LT339 W209A on GFP-FYVE dots (green). Quantification of the GFP-FYVE dots were counted from three independent experiments (CTR: n = 68; LT339: n = 38; LT339 W209A : n = 39). Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (H) The PIK3C3 kinase activity was evaluated in the FLAG IP lysates from cells expressing LT339 or LT339 W209A using the PI3K lipid kinase assay ( n = 4). (D, E, G and H) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant, one-way ANOVA with post-hoc Dunnett’s (D) or Tukey’s (G and H), and paired t -test (E).

Article Snippet: FLAG-tagged BECN1 full-length (BECN1 FL 24,388) and deletion mutants (BECN1 1–150, 24389; BECN1 151–241, 24390; BECN1 243–450, 24392; BECN1 Δ151–241, 24393) were purchased from Addgene (deposited by Qing Zhong).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Functional Assay, Expressing, Construct, Negative Control, Co-Immunoprecipitation Assay, Transfection, Staining, Kinase Assay

Tat-BECN1 peptide induces autophagy and cell death in vitro . (A) Three MCPyV + MCC cell lines were treated with various concentrations of Tat-BECN1 (T-B) or Tat-scrambled (T-S) for 6 h, followed by IB analysis of autophagy markers SQSTM1 and MAP1LC3B-II. TUBA1B, loading control. (B) Evaluation of cell viability by trypan blue assay in (A) (WaGa: n = 5; MKL-1: n = 5; MKL-2: n = 4). (C) Treatment of WaGa cells with 10 μM T-S or T-B, together with 3-methyladenine (3-MA, an autophagy inhibitor) and/or the pan-caspase inhibitor (PCI, an apoptosis inhibitor). Propidium iodide (pi)-positive flow cytometry assay ( n = 10), trypan-blue cell viability assay ( n = 7) and CellTiter-glo assay ( n = 4) were performed to evaluate cell viability upon different treatment conditions. (B and C) * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, were calculated by two-way ANOVA (B) or one-way ANOVA with post-hoc Bonferroni’s-test (C). Data represent mean ± SEM. (D and E) The interaction between BECN1 and its interactome was evaluated using co-IP and IB in (D) WaGa cells or (E) KIT-HEK293 cells co-transfected with FLAG-tagged BECN1 and LT339 after treatment with 10 μM of T-B or T-S for 4 h. IgG-IP was used as a negative control. The MAP1LC3B-II:TUBA1B ratios relative to T-S treatment in BECN1 IP are shown below the IB.

Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: Tat-BECN1 peptide induces autophagy and cell death in vitro . (A) Three MCPyV + MCC cell lines were treated with various concentrations of Tat-BECN1 (T-B) or Tat-scrambled (T-S) for 6 h, followed by IB analysis of autophagy markers SQSTM1 and MAP1LC3B-II. TUBA1B, loading control. (B) Evaluation of cell viability by trypan blue assay in (A) (WaGa: n = 5; MKL-1: n = 5; MKL-2: n = 4). (C) Treatment of WaGa cells with 10 μM T-S or T-B, together with 3-methyladenine (3-MA, an autophagy inhibitor) and/or the pan-caspase inhibitor (PCI, an apoptosis inhibitor). Propidium iodide (pi)-positive flow cytometry assay ( n = 10), trypan-blue cell viability assay ( n = 7) and CellTiter-glo assay ( n = 4) were performed to evaluate cell viability upon different treatment conditions. (B and C) * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, were calculated by two-way ANOVA (B) or one-way ANOVA with post-hoc Bonferroni’s-test (C). Data represent mean ± SEM. (D and E) The interaction between BECN1 and its interactome was evaluated using co-IP and IB in (D) WaGa cells or (E) KIT-HEK293 cells co-transfected with FLAG-tagged BECN1 and LT339 after treatment with 10 μM of T-B or T-S for 4 h. IgG-IP was used as a negative control. The MAP1LC3B-II:TUBA1B ratios relative to T-S treatment in BECN1 IP are shown below the IB.

Techniques: In Vitro, Control, Flow Cytometry, Viability Assay, Glo Assay, Co-Immunoprecipitation Assay, Transfection, Negative Control

Tat-BECN1 inhibits tumor growth in MCC xenograft. (A) Schematic illustration of the experimental design for the MCC xenograft model. (B) Tumor volume was measured every other day and calculated as stated in the method section. (C) Representative pictures of the xenograft tumors from mice treated with T-S or T-B after surgical excision. (D) The xenograft tumors were harvested and weighed at the end point ( n = 6 for each condition) (E) Left : representative images of MKI67 expression determined by IHC in xenograft tumors from mice treated with T-S or T-B. Scale bar: 40 µm. Right : quantification of MKI67 h-score in xenograft tumors ( n = 6 for each condition). Each filled circle represents the mean of MKI67 H-score within three randomly analyzed areas in each xenograft tumor. H-score was quantified using the QuPath software. (F) Representative images of MAP1LC3B expression determined by IHC in xenograft tumors from mice treated with T-S or T-B. Scale bar: 40 µm. (G) Top : immunoblots show the expressions of SQSTM1, MAP1LC3B and LT in xenograft tumors from the T-S and T-B treatment groups. Bottom : quantification of SQSTM1, MAP1LC3B-II and LT levels in xenograft tumors by IB ( n = 5). (H) Representative images show the ultrastructure of xenograft tumors analyzed by transmission electron microscopy. Autophagic vesicles are indicated as AP (autophagosomes, yellow arrowheads) and AL (autolysosomes, blue arrowheads). A total of 39 and 57 TEM images from two xenograft tumors in T-S and T-B treatment group, respectively, were analyzed. Insets refer to enlarged images of autophagic vesicles AP and AL. Scale bar: 3 μm in overview images and 300 nm in inset images. (B, D, E, G and H) Data represent mean ± SEM. * p < 0.05, ** p < 0.01, ns = not significant were assessed by two-way ANOVA (B), Mann-Whitney U -test (D, E and G), and unpaired t -test (H).

Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: Tat-BECN1 inhibits tumor growth in MCC xenograft. (A) Schematic illustration of the experimental design for the MCC xenograft model. (B) Tumor volume was measured every other day and calculated as stated in the method section. (C) Representative pictures of the xenograft tumors from mice treated with T-S or T-B after surgical excision. (D) The xenograft tumors were harvested and weighed at the end point ( n = 6 for each condition) (E) Left : representative images of MKI67 expression determined by IHC in xenograft tumors from mice treated with T-S or T-B. Scale bar: 40 µm. Right : quantification of MKI67 h-score in xenograft tumors ( n = 6 for each condition). Each filled circle represents the mean of MKI67 H-score within three randomly analyzed areas in each xenograft tumor. H-score was quantified using the QuPath software. (F) Representative images of MAP1LC3B expression determined by IHC in xenograft tumors from mice treated with T-S or T-B. Scale bar: 40 µm. (G) Top : immunoblots show the expressions of SQSTM1, MAP1LC3B and LT in xenograft tumors from the T-S and T-B treatment groups. Bottom : quantification of SQSTM1, MAP1LC3B-II and LT levels in xenograft tumors by IB ( n = 5). (H) Representative images show the ultrastructure of xenograft tumors analyzed by transmission electron microscopy. Autophagic vesicles are indicated as AP (autophagosomes, yellow arrowheads) and AL (autolysosomes, blue arrowheads). A total of 39 and 57 TEM images from two xenograft tumors in T-S and T-B treatment group, respectively, were analyzed. Insets refer to enlarged images of autophagic vesicles AP and AL. Scale bar: 3 μm in overview images and 300 nm in inset images. (B, D, E, G and H) Data represent mean ± SEM. * p < 0.05, ** p < 0.01, ns = not significant were assessed by two-way ANOVA (B), Mann-Whitney U -test (D, E and G), and unpaired t -test (H).

Techniques: Expressing, Software, Western Blot, Transmission Assay, Electron Microscopy, MANN-WHITNEY

Model of kit-mediated autophagy suppression by MCPyV LT in MCC. In normal cells, newly synthesized KIT proteins traffic from the endoplasmic reticulum (ER) to the golgi apparatus, where they undergo a series of glycosylations before being transported to the plasma membrane. After binding to its ligand KITLG, KIT is phosphorylated and rapidly internalized by endocytosis and then degraded by lysosomes or recycling to the cell surface. In MCPyV + MCC cells, the endocytosed KIT is blocked from degradation by the LT-VPS39 interaction, which leads to stabilization of KIT in the paranuclear compartment. The paranuclear KIT then interacts with BECN1 that enhances the interaction of BECN1 with BCL2 complex, leading to suppression of autophagy to support cell survival. MUR: MCPyV unique region of LT.

Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: Model of kit-mediated autophagy suppression by MCPyV LT in MCC. In normal cells, newly synthesized KIT proteins traffic from the endoplasmic reticulum (ER) to the golgi apparatus, where they undergo a series of glycosylations before being transported to the plasma membrane. After binding to its ligand KITLG, KIT is phosphorylated and rapidly internalized by endocytosis and then degraded by lysosomes or recycling to the cell surface. In MCPyV + MCC cells, the endocytosed KIT is blocked from degradation by the LT-VPS39 interaction, which leads to stabilization of KIT in the paranuclear compartment. The paranuclear KIT then interacts with BECN1 that enhances the interaction of BECN1 with BCL2 complex, leading to suppression of autophagy to support cell survival. MUR: MCPyV unique region of LT.

Techniques: Synthesized, Clinical Proteomics, Membrane, Binding Assay

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flag becn1 24388 plasmids (Addgene inc)

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