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Image Search Results
Journal: Developmental cell
Article Title: Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection.
doi: 10.1016/j.devcel.2023.08.013
Figure Lengend Snippet: Figure 6. Usp25 interacts with and stabilizes the Erlin1/2 complex (A) FLAG-Usp25 expressed in A549 cells were either mock infected or H1N1pdm-infected (MOI = 1). Usp25 was immunoprecipitated from mock and H1N1pdm- infected cells on anti-FLAG M2 affinity beads and trypsin digested to identify interactors using mass spectrometry. Volcano plots indicate protein enrichments in mock and H1N1pdm-infected cells. (B) Candidates with the highest number of unique peptides were isolated specifically from H1N1pdm-infected cells. (C) A549 cells expressing either FLAG-tag or FLAG-Usp25WT were either mock infected or H1N1pdm infected. Expression of Erlin1 and Erlin2 were measured by immunoblotting. Gapdh levels measured as loading control. A549 cells either Usp25/, or expressing Usp25WT or Usp25C178S were mock or H1N1pdm infected, and Erlin2 expression measured by immunoblotting. (D and E) IAV-infected A549 cells either Usp25/ or expressing Usp25WT or Usp25C178S were pulsed with [35S]cysteine/methionine and chased for indicated time intervals. At each time point, Erlin1/2 was immunoprecipitated, resolved by SDS-PAGE and detected by autoradiography. (F) HEK-293T cell extracts, transiently co-transfected with Myc-Usp25 together with the indicated plasmids encoding FLAG-IFIT1, FLAG-IFIT5, influenza FLAG-M2, and FLAG-M2 F91S (containing a mutation in its LC3-interacting region to prevent association with autophagosomes) were immunoprecipitated on anti-FLAG M2 affinity beads. The immunoprecipitates were analyzed by immunoblotting with anti-Myc to validate their interaction with Usp25. Transfected FLAG- tagged proteins were detected by immunoblotting the cell lysates (CL) with anti-FLAG. Gapdh was used as loading control. (G) A549 cells expressing either myc-BirA or myc-BirA-Usp25 cultured in biotin-supplemented media were either mock or H1N1pdm-infected. Lysates were captured on Neutravidin beads and eluates were immunoblotted for Erlin1 and Erlin2. (H and I) IAV-infected WT and Usp25/ cells were pulsed with [35S]cysteine/methionine for 10 min and chased in cold media for indicated time intervals in DMSO alone or 50 mM MG132-treated (o/n) cells. Erlin1 and Erlin2 were immunoprecipitated, resolved by SDS-PAGE and detected by autoradiography. Quantitation was performed as relative amounts normalized to the starting amount at t = 0. See also Figure S6.
Article Snippet: Vero ATCC CCL-81 Oligonucleotides GGCACCAAGGCACATAACGG (sgRNA) This study N/A GAGACTGAAAGATTACCTCA (sgRNA) This study N/A AGCAAAAGCAGG (uni-12) This study N/A AGTAGAAACAAGG (uni-13) This study N/A GACCAATCCTGTCACCTCTGA (M Gene-Forward) This study N/A AGGGCATTTTGGACAAAGCGTCTA (M Gene-Reverse) This study N/A CACCATTGGCAATGAGCGGTTC (b-actin-Forward) This study N/A AGGTCTTTGCGGATGTCCACGT (b-actin-Reverse) This study N/A Recombinant DNA pSpCas9(BB)-2A-Puro (PX459) V2.0 Addgene Addgene plasmid #62988 Usp25-PX459 This study N/A Flag-tagged-Usp25 Xu et al.49 N/A Flag-tagged-Usp25 (C178S) Xu et al.49 N/A Myc-tagged- RIG I te Velthuis et al.50 N/A Flag-tagged-M2 This study N/A 3xFlag IFIT1 Addgene Addgene plasmid #53554
Techniques: Infection, Immunoprecipitation, Mass Spectrometry, Isolation, Expressing, FLAG-tag, Western Blot, Control, SDS Page, Autoradiography, Transfection, Mutagenesis, Cell Culture, Quantitation Assay
Journal: International Journal of Molecular Sciences
Article Title: Gene Therapy Approach for Treatment of Obese Agouti Mice
doi: 10.3390/ijms252212144
Figure Lengend Snippet: Schematic representation of plasmid map pAAV-FoxP4, pAAV-PRDM16, and pAAV-FST.
Article Snippet: The mouse PRDM16 gene was amplified from a commercially available plasmid,
Techniques: Plasmid Preparation
Journal: International Journal of Molecular Sciences
Article Title: Gene Therapy Approach for Treatment of Obese Agouti Mice
doi: 10.3390/ijms252212144
Figure Lengend Snippet: Progression of body weight change relative to weight before AAV administration in agouti mice treated at 12 weeks of age with an intra-WAT injection of empty AAV (Control), AAV-FoxP4, AAV-PRDM16, or AAV-FST vectors. Data are presented as mean ± S.D. values of three mice for each group. * p < 0.1 control group vs. AAV-FoxP4 group, # p < 0.1 control group vs. AAV-PRDM16 group, † p < 0.1 control group vs. AAV-FST group.
Article Snippet: The mouse PRDM16 gene was amplified from a commercially available plasmid,
Techniques: Injection, Control
15 ]. Lipids showing p -value > 0.05 are marked «Ns» (non-significant)." width="100%" height="100%">
Journal: International Journal of Molecular Sciences
Article Title: Gene Therapy Approach for Treatment of Obese Agouti Mice
doi: 10.3390/ijms252212144
Figure Lengend Snippet: The fold changes of the identified lipids between the AAV9-FST, AAV8-FoxP4, and AAV8-PRDM16 groups against the control (empty AAV). Fold change and p -value were calculated in Metaboanalyst 5.0 [
Article Snippet: The mouse PRDM16 gene was amplified from a commercially available plasmid,
Techniques: Control