dsrnas from drosophila rnai library (open biosystem)  (Thermo Fisher)

Journal: Genes & Development

Article Title: Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis

doi: 10.1101/gad.351856.124

Figure Lengend Snippet: Identification of Hyd as a critical E3 ubiquitin ligase mediating Lin-induced Bowl degradation. ( A ) Schematic diagram of a pooled RNAi screen for E3 ligases required for Lin-induced Bowl degradation. S2R + cells transfected with HA-Bowl and FLAG-Lin were split into 24 well plates, each well treated with a pool of dsRNAs targeting four different E3 ligases, followed by Western blot analysis of cell lysates. ( B ) Confirmation of Hyd as an E3 ligase required for Lin-induced Bowl degradation. S2R + cells expressing HA-Bowl and FLAG-Lin were treated with different Hyd dsRNAs. (Hyd-1) dsRNA in the original RNAi library (Hyd-2, Hyd-3, and Hyd-4) three synthesized dsRNAs targeting different regions of Hyd. HA-Bowl was normally undetectable under such conditions due to Lin-induced degradation. However, it was stabilized by RNAi against Hyd or Lin. GFP dsRNA was included as a negative control. ( C ) S2R + cells expressing the indicated constructs were subjected to coimmunoprecipitation (co-IP) assay as indicated. Cells were treated with 10 μM PS-341 for 4 h before harvesting. Interaction was readily detected between FLAG-Lin and HA-Bowl (lane 3 ) and between FLAG-Lin and Hyd-HA (lane 4 ). (Lane 5 ) Neither pairwise interaction was affected by coexpression of the third protein. ( D ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Cells were treated with PS-341 as in C . Co-IP between FLAG-Bowl and Hyd-HA was detected only in the presence of Myc-Lin (cf. lanes 2 and 5 ). ( E ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. (HA-Bowl 122–373K137R ) The K137R mutant form of Bowl fragment 122–373 as described in C. Note that the Lin–Bowl co-IP was severely impaired by lin A25 , but not lin A24 , mutation. ( F ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Note that the Lin–Hyd co-IP was impaired by both lin A24 and lin A25 mutations. ( G ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Cells were treated with PS-341 as in C . Note the disruption of the Lin–Bowl interaction by Drm (cf. lanes 2 and 3 ). ( H ) S2R + cells expressing the indicated constructs were treated with PS-341 before IP with FLAG antibody. The IP product was probed with antiubiquitin antibody to detect Bowl ubiquitination. Lin-induced Bowl ubiquitination was suppressed by Drm. ( I ) Schematic model for the regulation of Bowl degradation. In the absence of Drm, Lin promotes Bowl degradation by functioning as a substrate adaptor protein recruiting Bowl to Hyd. Drm competes with Bowl for Lin binding, thus releasing Bowl from Lin–Hyd, resulting in Bowl stabilization.

Article Snippet: For the dsRNA screen, the DNA templates of dsRNAs from Drosophila RNAi library (Open Biosystem) were PCR-amplified and transcribed with 5XMEGAScript T7 kit (Ambion).

Techniques: Transfection, Western Blot, Expressing, Synthesized, Negative Control, Construct, Co-Immunoprecipitation Assay, Mutagenesis, Disruption, Binding Assay

Journal: Genome Biology

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

doi: 10.1186/gb-2011-12-10-r104

Figure Lengend Snippet: PCR amplification, quantitative PCR and Illumina sequencing schema . (a) Diagrammatic representation of the complete integrated shRNA construct. LTR, long terminal repeat; Ze, zeomycin resistance bacterial selectable marker; tGFP, turbo GFP; IRES, internal ribosome entry site; Puro, puromycin mammalian selectable marker; RRE, Rev response element; sinLT, self-interacting LTR. (b) The structure of the shRNAmir construct. The sense and antisense shRNA sequences hybridize to form a hairpin loop structure. (c) PCR primer alignment to the shRNA construct. The PCR primers incorporate p7 and p5 sequences to enable capture on an Illumina flowcell. (d) Sequencing primer, quantitative PCR (qPCR) primer and qPCR dual label probe alignment to the shRNA PCR product. CMV, cytomegalovirus; DLP, dual-labeled probe.

Article Snippet: Although the following methods are suitable for most viral shRNA libraries, the work described here used the Thermo Scientific Open Biosystems GIPZ Lentiviral human shRNAmir library (version 2).

Techniques: Amplification, Real-time Polymerase Chain Reaction, Sequencing, shRNA, Construct, Marker, Labeling

Journal: Cell reports

Article Title: Acetate Promotes T Cell Effector Function during Glucose Restriction

doi: 10.1016/j.celrep.2019.04.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD8α Biolegend Clone 53–6.7 Anti-mouse CD44 Biolegend Clone IM7 Anti-mouse CD45.1 Biolegend Clone A20 Anti-mouse CD45.2 Biolegend Clone 104 Anti-mouse PD-1 Invitrogen Clone J43 Anti-mouse CD25 Biolegend Clone 3C7 Anti-mouse CD69 Biolegend Clone H1.2F3 Anti-mouse CD62L Biolegend Clone MEL-14 Anti-mouse CD71 Biolegend Clone RI7217 Anti-mouse Granzyme B eBioscience Clone NGZB Anti-mouse IFN-γ Biolegend Clone XMG1.2 Anti-GFP Biolegend Clone FM264G Anti-mouse α Tubulin Cell Signaling Clone 11H10 Anti-mouse ACSS2 Thermo Scientific Cat. PA5–26612 Anti-mouse MCT-4 Santa Cruz Clone H-90 sc-50329 Anti-mouse MCT-1 Santa Cruz Clone T-19 sc-14917 Anti-mouse H3 Cell Signaling Cat. 9715 Anti-mouse H4 Cell Signaling Cat. 13919 Anti-mouse acetylated H3 Millipore Cat. 06–599 Anti-mouse acetylated H4 Millipore Cat. 06–866 Anti-mouse acetylated H3K9/14 Diagenode Cat. C15410200 Anti-mouse acetylated H3K27 Diagenode Cat. C15410196 Bacterial and Virus Strains Ctrl hairpin Open Biosystems MSCV-LTRmiR30-PIG (LMP) Addgene plasmid #24071 Acss1 hairpin (same backbone as Ctrl hairpin and Acss1 sequence) Open Biosystems MSCV-LTRmiR30-PIG (LMP) Addgene plasmid #24071 Acss2 hairpin (same backbone as Ctrl hairpin and Acss2 sequence) Open Biosystems MSCV-LTRmiR30-PIG (LMP) Addgene plasmid #24071 Ctrl MigR1 Gift from Steve Reiner MIGR1 plasmid #27490 Acss2 MigR1 Dharmacon MGC Mouse Acss2 cDNA(Clone ID: 6515568) Biological Samples HCV-infected blood Uniklinik Freiburg (Dr. Bertram Bengsch) NA Chemicals, Peptides, and Recombinant Proteins SIINFEKL peptide New England Peptide Cat. BP10–915 FBS GIBCO Lot.

Techniques: Plasmid Preparation, Sequencing, Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software