flag control  (Addgene inc)

Journal: Cell Death Discovery

Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression

doi: 10.1038/s41420-025-02717-0

Figure Lengend Snippet: A WT or G2E3-KO AsPC-1 cells were transfected with control plasmid or plasmids encoding G2E3-FLAG. The cells were then analyzed by SDS-PAGE and immunoblotting with antibodies to the indicate proteins. B Quantification of ratio of LC3B-II, GABARAP and GABARAPL1 to Vinculin. The indicated p-values were calculated using means of three independent experiments ±SEM. n.s., not significant, * p < 0.05, ** p < 0.01. Significance was calculated with Student’s t test. C HEK293TN WT and AsPC-1 WT were transfected with plasmids encoding G2E3-FLAG or control plasmid FLAG. After 48 h, cell lysates and immunoprecipitates were analyzed using SDS-PAGE and immunoblotting with corresponding antibodies. D Immunofluorescent staining of endogenous GABARAP, GABARAPL1, LC3B, and exogenous G2E3 in G2E3 KO AsPC-1cells. G2E3KO AsPC-1 cells were transfected with plasmids encoding G2E3-FLAG. After 48 h, cells were fixed. G2E3-FLAG was stained with anti-FLAG antibody and the co-localization of the endogenous GABARAP, LC3B and GABARAPL1 were visualized under confocal microscope. Scale bar: 10 μm.

Article Snippet: pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological.

Techniques: Transfection, Control, Plasmid Preparation, SDS Page, Western Blot, Staining, Microscopy

Journal: Cell Death Discovery

Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression

doi: 10.1038/s41420-025-02717-0

Figure Lengend Snippet: A WT or G2E3-KO AsPC-1 cells were fixed and stained with Lamp1 (red), LC3 (green), and DAPI (blue). Scale bar 10 μm. B The Pearson’s coefficients of LC3B overlap with Lamp1 in ( A ) were quantified using Fiji software. Mean ± SEM, n = 10. **** p < 0.0001. Significance was calculated with Student’s t test. C Co-localization analysis of endogenous Lamp1 and exogenously expressed G2E3-FLAG. Line plots exemplify degree of co-localization of G2E3 and Lamp1 signals.

Article Snippet: pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological.

Techniques: Staining, Software