prl tk cxcr4 6x reporter plasmid  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF interacts with eIF4E at the m 7 G cap, independent of intact eIF4G1. ( a ) Reporter assay quantifying the relative Renilla luciferase expression from the indicated 3′ UTR luciferase reporters in untreated and TGF-β-treated MCF-10A cells. Data were normalized to Firefly luciferase expression and are presented as fold change of this normalized signal relative to CXCR4 reporter in untreated MCF-10A cells. ( b ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of indicated Renilla luciferase reporters (pRL-TK) in untreated and TGF-β-treated MCF-10A cells. Data were normalized to endogenous ACTB expression. ( c ) As in (a), with the indicated 3′ UTR luciferase reporters driven from an EMCV internal ribosomal entry site (IRES) in untreated and TGF-β-treated MCF-10A cells. ( d ) As in (b), for reporter assays in panel (c). ( e ) Right six lanes—immunoblots of indicated immunoprecipitates from whole-cell lysates derived from MCF-10A cells treated with TGF-β for 72 h. One half of each total immunoprecipitate was digested with RNase A prior to immunoblotting with the indicated antibodies. Right six lanes—as in the left six lanes, but lysates were digested with coxsackievirus 2A protease to cleave eIF4G1 before immunoprecipitation. CT = C-terminal; FL = full length; NT = N-terminal. ( f ) m 7 GTP cap analog binding assays utilizing cytosolic extracts derived from MCF-10A cells treated with TGF-β for 72 h. As above, one half of each extract was digested with coxsackievirus 2A protease to cleave eIF4G1 before the assay. ( g ) Proximity ligation assays using the indicated pairs of antibodies on MCF-10A cells treated with TGF-β for 72 h. In all panels, results are representative of at least three independent experiments and error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 (Student’s t-test).

Article Snippet: WT or GRE deletion mutant CRLF1 and SNAI1 3′ UTRs were fused downstream of the Renilla luciferase coding sequence in the broadly used pRL-TK CXCR4 6x reporter plasmid ([ ], Plasmid #11 308, Addgene, Cambridge, MA) as described before [ ].

Techniques: Reporter Assay, Luciferase, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Derivative Assay, Immunoprecipitation, Binding Assay, Ligation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 stimulates translation of GRE-containing EMT effector mRNAs in the context of reduced eIF4G1 function. ( a ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using anti-CELF1, anti-eIF4E, and anti-eIF4G1 antibodies or mouse and rabbit IgG. ACTB and GAPDH are non-GRE-containing negative control mRNAs. ( b ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using tandem anti-eIF4E/anti-CELF1 immunoprecipitation, tandem anti-eIF4E/anti-eIF4G1 immunoprecipitation, or tandem immunoprecipitation with mouse and rabbit IgGs. ACTB and GAPDH are non-GRE-containing negative controls. ( c, d ) Efficiency of in vitro translation of indicated capped and polyadenylated Renilla luciferase reporter mRNAs in mock or 2A protease-digested cell-free extract. ( e, f ) Efficiency of in vitro translation of reporter mRNAs as described in panels (c) and (d), but with mock-depleted ( Beads ) cell-free extract, eIF4G1-immunodepleted ( ID ) cell-free extract, or eIF4G1-immunodepleted cell-free extract reconstituted by addition of 20 nM enriched eIF4G1 and/or an equivalent concentration of recombinant CELF1. In panels (c–f), all extracts were derived from TGF-β-treated MCF-10A cells transiently transfected with shRNAs targeting either GLB1 (c, e) or CELF1 (d, f). CXCR4 = control, WT = wild-type 3′ UTR, ΔGRE =3′ UTR with deletion of GRE. In all panels, results are representative of at least three independent experiments. Error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 ( a, b, e, f : ANOVA with Dunnet’s post-hoc test; c, d : Student’s t-test).

Article Snippet: WT or GRE deletion mutant CRLF1 and SNAI1 3′ UTRs were fused downstream of the Renilla luciferase coding sequence in the broadly used pRL-TK CXCR4 6x reporter plasmid ([ ], Plasmid #11 308, Addgene, Cambridge, MA) as described before [ ].

Techniques: Cross-linking Immunoprecipitation, Quantitative RT-PCR, Negative Control, Immunoprecipitation, In Vitro, Luciferase, Concentration Assay, Recombinant, Derivative Assay, Transfection, Control, Standard Deviation