Journal: Nucleic Acids Research

Article Title: Efficient suppression of premature termination codons with alanine by engineered chimeric pyrrolysine tRNAs

doi: 10.1093/nar/gkae1048

Figure Lengend Snippet: ( A ) Schematic representation of the plasmid system used to measure PTC suppression in BRCA1 . Human BRCA1 (residues 1396–1863) containing an in-frame UAG PTC was expressed as a genetic fusion to the Gal4 DNA-binding domain (Gal4DBD). Three copies of tRNA V2.1 were expressed from the same plasmid. The Gal4DBD-BRCA1 plasmid was co-transfected with a plasmid containing the firefly luciferase (Fluc) gene and five copies of the Gal4 upstream activation sequence (5× UAS). In this assay, Fluc transcription is dependent on BRCA1-mediated transactivation. ( B ) BRCA1 activity in HEK 293 cells expressing BRCA1 with a UAG PTC at the indicated position with (+) and without (–) co-expression of tRNA V2.1 . Data are displayed as the mean ± SEM of three biological replicates. * P < 0.05, ** P < 0.005, unpaired student’s t -test.

Article Snippet: Briefly, the yeast Gal4 DNA-binding domain (Gal4; residues 1–100) was cloned from plasmid Gal4-VP16 (a gift from Lea Sistonen; Addgene plasmid # 71728; http//n2t.net/addgene:71728 ; RRID:Addgene_71728) as a genetic fusion to human BRCA1 (residues 1396–1863) (a gift from Stephen Elledge; Addgene plasmid #14999; http://n2t.net/addgene:14999 ; RRID:Addgene_14999) in a pCDNA3.1 plasmid harboring three copies of the gene encoding tRNA V2.1 under control of a 7SK promoter.

Techniques: Plasmid Preparation, Binding Assay, Transfection, Luciferase, Activation Assay, Sequencing, Activity Assay, Expressing

Journal:

Article Title: UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans

doi:

Figure Lengend Snippet: UNC-4 and UNC-37 interact in yeast two-hybrid assays. UNC-4 proteins are fused to the GAL4 DNA-binding domain; UNC-37 proteins are fused to the GAL4 activation domain. Assays for growth on His− plates were performed in the presence or absence of 3-AT: (+) growth on 100 mm 3-AT; (−) weak or no growth on 100 mm 3-AT. (A) Interactions between full-length UNC-37 and UNC-4 deletion mutants. UNC-4 refers to the full-length protein; UNC-4C represents the carboxy-terminal truncated protein (152–252). Other UNC-4 truncations are denoted by amino acid boundaries. (B) Interactions of UNC-37 and UNC-37(E580K) with UNC-4 eh1 region missense mutations. The allele numbers of previously identified unc-4 mutations are indicated. Asterisks indicate relative positions of mutations. (C) Selected growth assays ± 100 mm 3-AT from A and B.

Article Snippet: Mutant UNC-4 fusion protein stability was analyzed by Western blots of yeast protein extracts using an anti-GAL4 DNA-binding domain polyclonal antibody (Santa Cruz, Inc.) All UNC-4 fusion proteins were stably expressed (data not shown).

Techniques: Binding Assay, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: AIPL1, a protein implicated in Leber's congenital amaurosis, interacts with and aids in processing of farnesylated proteins

doi: 10.1073/pnas.2134194100

Figure Lengend Snippet: Interaction of AIPL1 and AIPL1 mutants with farnesylated proteins in vivo. Yeast (PJ69-4) containing the indicated bait fused to the GAL4-activation domain and preys fused to GAL4-DNA-binding domain were tested for their ability to promote growth of yeast on synthetic media lacking leucine, tryptophan, histidine, and adenine (LTHA) (–). As a control, the same combination was grown in medium lacking leucine and tryptophan, but containing histidine and adenine (+). (a) Interaction between AIPL1 and DNAJA2 is specific. Yeast containing bait AIPL1 or AIPL1 lacking the C-terminal 56 amino acids (ΔCT-AIPL1) and prey DNAJA2 grow on LTHA media (–). (b) AIPL1 specifically interacts with proteins that contain a conserved C-terminal farnesylation signal. AIPL1 does not interact with farnesylation-defective mutant forms of DNAJA2 (–SAHQ) or γ-transducin (–SVIS). AIPL1 does not interact with geranylgeranylated forms of DNAJA2 (–CAHL) or γ-transducin (–CVIL) mutants. (c) AIPL1 does not interact with AhR. Yeast containing AIPL1 or AIP as bait and AhR as prey were tested for the ability to grow on LTHA media (–). (d) AIP does not interact with farnesylated proteins. (e) Farnesylation of target proteins is necessary for interaction with AIPL1 in yeast. Yeast (PJ69-4) lacking the farnesyltransferase gene (ΔRam1) were tested for their ability to support the interaction of AIPL1 with DNAJA2 and γ-transducin. As a control, interaction of AIPL1 with FLJ13855 and AIP with AhR was tested.

Article Snippet: Yeast GAL4 DNA-binding domain fusions were detected by using HRP-linked Anti-GAL4 (RK5C1) (Santa Cruz Biotechnology), and His-tagged proteins were detected by HRP-linked INDIA His probe (Pierce).

Techniques: In Vivo, Activation Assay, Binding Assay, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 activates AP-1-dependent promoters by inducing c-Jun transactivation independent of FHL2. (A) Protein levels of FHL2 and p57 in different cell lines. 100 μg of total protein extract were analyzed by immunoblotting for FHL2 and p57 using a mouse monoclonal FHL2- and a rabbit polyclonal p57-specific antibody. CEM, human leukemic; HEL, human erythroleukemic; HL-1, mouse cardiac muscle; WM35, human melanoma; 293, human embryonic kidney; MCF-7, human breast cancer; HRT-18, human colon cancer; U2OS, human osteosarcoma; HeLa, human cervix carcinoma. The unspecific band at 70 kD ( ∗ ) after probing for p57 was used to judge equal loading. (B) Knockdown of FHL2 using four different doxycyclin-inducible shRNAs in stable pools of lentivirally transduced HRT-18 cells. Western blot analysis of extracts obtained from HRT-18 cells. The expression of shRNAs was induced by the addition of doxycycline (1 μg/mL) for 72 h. Cells expressing shRNA targeting luciferase mRNA (shLuc) served as a control. The FHL2-specific antibody revealed efficient (sh215, sh718) and less efficient (sh428, sh598) inhibition of FHL2 expression. GAPDH served as a loading control. (C) Regulation of an artificial AP-1-dependent promotor (schematic representation top panel) by p57. HRT-18.sh215 cells either untreated (– Doxy) or treated with 1 μg/mL doxycycline (+ Doxy) for 72 hours were transfected with 5xTRE-Luc reporter gene construct and 10 ng pUbi-Rluc (expressing renilla luciferase as a transfection control) together with the p57 expression vectors as indicated. A total of 48 h after transfection, cells were harvested and a Dual luciferase assays performed. Relative promoter activities were expressed as firefly luciferase activities normalized for renilla-luciferase activities. Shown are the mean values ± SD from six independent experiments. (D) Gal4-dependent luciferase reporter gene experiments comparing the effect of p57 on c-Jun and c-Fos transactivation. 293 cells with stably integrated Gal4-dependent luciferase reporter gene (293FR) were transfected with DNA-expression plasmids for Gal-DBD, Gal-Jun or Gal-Fos and p57. Schematic representation of the reporter construct is shown in the top panel. In addition, 10 ng pUbi-Rluc expressing renilla luciferase were cotransfected as control. A total of 30 h after transfection, cells were extracted and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (–) for Gal-Jun and Gal-Fos which are set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 15, top: 100 to 2,250). Shown is the mean ± SD from three independent experiments. (E) Gal4-dependent luciferase reporter gene experiments analyzing effect of p57 on c-Jun transactivation. HRT-18 cells with stably integrated Gal4-dependent luciferase reporter gene (HRT-18FR) were transfected with DNA-expression plasmids for Gal-Jun, p57 and the indicated shRNAs. pUbi-Rluc (expressing renilla luciferase) was cotranfected as a transfection control. shco was used as a control shRNA not targeting any known mRNA. 72 hours after transfection, cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (“–”/“shco”) cells which is set to 1. Shown is the mean ± SD from three independent experiments. Level of significance is indicated. ** = Highly significant, n.s. = not significant.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Western Blot, Expressing, shRNA, Luciferase, Inhibition, Transfection, Construct, Stable Transfection, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: Strong activation of Gal-Jun activity is specific for p57 and requires both, the N -terminal and C -terminal domains of p57. (A) Gal4-dependent luciferase reporter gene experiments comparing the effect of the Cip/Kip-family members p57, p27, and p21 on c-Jun transactivation. 293FR cells were cotransfected with DNA-expression plasmids for Gal-DBD, Gal-Jun and p57, p27 or p21. pUbi-Rluc (expressing renilla luciferase) served as a transfection control. A total of 30 h after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. To represent minor activation, the y-axis is split into two segments (bottom: 0 to 15, top: 100 to 1,500). Shown is the mean ± SD from three independent experiments. (B) Similar experiment to (A) comparing p57 and cyclin/CDK-binding deficient p57 mutant. 293FR cells were cotransfected with DNA-expression plasmids for Gal-DBD, Gal-Jun and p57 or the cyclin/CDK-binding deficient p57-CK- mutant (CK-) and pUbi-Rluc. A total of 30 h after transfection, cells were harvested and subjected to dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. Shown is the mean ± SD from four independent experiments. (C) Schematic representation of p57 and its N -terminal and C -terminal domains. Key structural or functional regions of human p57 are indicated. Numbers above indicate positions of amino acid flanking distinct regions. Amino acid including the N -terminal (p57-Nt) and C -terminal (p57-Ct) domains are shown in brackets. (D) Experiment as described above (A) analyzing the contribution of p57-domains to the activation of Gal-Jun by p57. 293FR cells were cotransfected with DNA-expression plasmids for Gal-Jun and p57 or the indicated p57 domain mutants. A total of 30 hours after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 100, top: 100 to 1,500). Shown is the mean ± SD from three independent experiments.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Activation Assay, Activity Assay, Luciferase, Expressing, Transfection, Binding Assay, Mutagenesis, Functional Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: JNK-independent activation of c-Jun by p57. (A) Schematic of human wildtype c-Jun, Gal-Jun and Gal-Jun4A mutant. Functional domains and phosphorylation sites in the transactivation domain (TAD) are indicated. A Gal4-DNA binding domain (DBD) fusion in frame to the aminoterminus of c-Jun or mutant c-Jun4A ranging from amino acids 1–223 is used in reporter gene experiments. (B) Gal4-dependent luciferase reporter gene experiments analyzing the role of the four major phosphorylation sites in the TAD of c-Jun in the activation by p57. 293FR cells were cotransfected with DNA-expression plasmids for Gal-Jun or the TAD phosphosite mutant Gal-Jun4A in absence or presence of p57 or the constitutive active upstream kinase of JNKs (ΔMEKK). pUbi-Rluc (expressing renilla luciferase) served as a transfection control. A total of 30 h after transfection cells were harvested and subjected to dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) Gal-Jun which was set to 1. For showing lower fold activations, y-axis is split into two segments (bottom: 0 to 2.5, top: 50 to 2,000). Shown are the results from three independent experiments. (C) p57 does not induce serine 63 or serine 73 phosphorylation of c-Jun. Immunoblot-analysis investigating the effect of p57 on c-Jun TAD-phosphorylation. 293 cells were transfected with expression vectors for c-Jun and p57 (both HA-tagged) or the constitutive active kinase ΔMEKK. Extracts were prepared 40 h after transfection and subjected to immunoblot-analysis by the LiCor Odyssey system to visualize expression of HA-Jun and Ser 63 and Ser 73 phosphorylation simultaneously. Mixture of Ser 63 and Ser 73 phospho-specific rabbit polyclonal and anti HA mouse monoclonal antibodies were used as first antibodies followed by a mixture of goat derived secondary antibodies coupled with fluorochromes for 680 (anti mouse) or 780 nm (anti rabbit). Three images are shown. Upper: 700 nm channel, phospho-Ser 63/73; middle: 800 nm channel, HA; lower: merged pseudo-colors from channel 700 (red) and 800 (green). Note that molecular weight of HA-Jun is shifted in the presence of the constitutive active kinase ΔMEKK and appears yellow in the merged image.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Activation Assay, Mutagenesis, Functional Assay, Binding Assay, Luciferase, Expressing, Transfection, Western Blot, Derivative Assay, Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57 Kip2

doi: 10.3389/fcell.2021.664609

Figure Lengend Snippet: p57 might recruit transcription repressors and activators to c-Jun. (A) Gal4-dependent luciferase reporter gene experiments analyzing the effect of p57-domain coexpression on p57-induced c-Jun-activation. Coexpression of p57-Ct may titrate out activators and coexpression of p57-Nt may titrate out repressors of c-Jun activation by p57. 293FR cells were cotransfected with expression plasmids for Gal-Jun, pUbi-Rluc, and p57 wildtype together with vectors expressing p57-Ct or p57-Nt or the related aminoterminal CDK-inhibitory domain of p27 (p27-Nt). A total of 30 h after transfection cells were harvested and subjected to Dual luciferase assays. Renilla-normalized values are expressed as fold activation relative to control transfected (Co.) which is set to 1. Shown is the mean ± SD from three independent experiments. (B) Mechanistic models for p57 in stimulating Gal-Jun transactivation function and a schematic of a putative transcription complex. (C) Model how p57 and FHL2 might act at endogenous promoters at AP-1 sites (TRE). Question mark in (B) and (C) indicates that there is no experimental proof for a direct interaction of p57 with the depicted proteins. For details see text.

Article Snippet: See for sequence information and cloning procedure. pcDNA3-FLAG-Fos WT was a gift from John Blenis (Addgene plasmid # 8966; http://n2t.net/addgene:8966 ; Addgene_8966 ) and is described elsewhere ( ). pSG424-Gal-c-fos expresses a fusion protein of the yeast transcription factor GAL4 DNA binding domain (aa 1–147) fused to rat c-Fos (aa 213–380) where DNA-binding and dimerization domains are absent and cloned by PCR-amplification of the corresponding codon using pcDNA3-FLAG-Fos WT as a template (oligonucleotide sequences, see ). pFC-MEKK (Agilent) expresses constitutive active MEKK (aa 380–672) which activates JNK.

Techniques: Luciferase, Activation Assay, Expressing, Transfection