pcep4 egfp drd3  (Addgene inc)

Journal: Mobile DNA

Article Title: Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis

doi: 10.1186/s13100-018-0138-z

Figure Lengend Snippet: Cytoplasmic localization of L1 ORF1 protein. Stress granule marker proteins TIA1 ( a , b ) and eIF3η ( c , d ) show minimal colocalization with endogenous ORF1p in cytoplasmic granules (shown by arrows) of unstressed human embryonal carcinoma 2102Ep cells ( a , c ) but colocalize in large stress granules of cells treated with Na-arsenite (0.5 mM for 1 hour) ( b , d ). Cell nuclei are stained with Hoechst. Size bars are 10 μm. e ORF1p cytoplasmic granules retain integrity during cellular mitosis. Patient sera-derived α-ANA-N marks nucleoli . Endogenous ORF1p is mostly excluded from metaphase chromatin plates (arrow), as shown by Hoechst staining (see arrow). f Ectopically expressed EGFP-tagged human ORF1p induces prominent cytoplasmic granules, but ( g ) deletion of its Q-N-rich region abolishes granule formation. h The ORF1p R159H point mutation reduces cytoplasmic granule formation by 50%. Approximately 500 cells were examined. i The R159H mutation abolishes cell culture LINE-1 retrotansposition. pc6-RPS-EGFP-ΔCMV wild-type or R159H mutant retrotransposition reporter constructs were transfected in HEK 293T cells and 5 days later the percentages of EGFP-positive cells were determined by flow cytometry. The construct 99-PUR-JM111-EGFP served as a negative control for retrotransposition . Each construct was tested in quadruplicate wells with results for one biological replicate shown

Article Snippet: 99-PUR-RPS-EGFP includes full-length L1-RP with the EGFP reporter cassette in its 3' UTR cloned in a modified version of pCEP4 vector (Invitrogen) lacking a cytomegalovirus (CMV) promoter.

Techniques: Marker, Staining, Derivative Assay, Mutagenesis, Cell Culture, Construct, Transfection, Flow Cytometry, Negative Control

Journal: Mobile DNA

Article Title: Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis

doi: 10.1186/s13100-018-0138-z

Figure Lengend Snippet: Increasing expression of some ALS-associated proteins alters retrotransposition in cell culture assays. n=number of biological replicates. a L1-RP is retrotransposition-competent in human SH-SY5Y neuroblastoma and mouse NSC-34 motor neuron-like cells. Cells in 6-well plates were infected with A/RT-pgk-L1RP-EGFP (Ad-L1) L1-reporter adenovirus at about 8 × 10 12 viral particles/ml. Flow cytometry analysis was performed at 9 days post-infection. b 99-PUR-RPS-EGFP was co-transfected in HEK 293T cells with empty vector (pcDNA3) or test constructs expressing tagged ALS-related proteins. Five days later, percentages of EGFP-positive cells were determined by flow cytometry. Each plasmid pair was transfected in four replicate wells, with at least 3 replicate experiments performed for each construct. Results are normalized to pcDNA3 vector control (lighter bar). Statistical significances compared with vector control were calculated by Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001). All test proteins were expressed as confirmed by Western blotting of whole cell lysates using α-DYKDDDDK (FLAG)-tag, α-Myc-tag, or α-V5-tag antibodies as indicated (top). Four-fold less V5-SQSTM1- and three-fold more V5-TBK1-transfected lysates were loaded on the gel. Full-length TBK1 expressed poorly, most of the protein existing as a high molecular weight smear. c FLAG-tagged TDP-43 co-immunoprecipitates T7-tagged L1 ORF1p complexes from HEK 293T cell lysates after α-FLAG-M2 affinity gel purification. Interaction is lost following treatment of lysates with RNase. d Expression of V5- or Myc-tagged TDP-43 strongly inhibits mouse IAP element retrotransposition in HeLa-JVM cell culture. Cells were treated with neomycin (G418) to select for retrotransposition events. Colony counts are not normalized. On the right are representative T 75 flask images with Giemsa-stained IAP retrotransposition-positive colonies in the absence or presence of TDP-43. The apparent diminished effect on retrotransposition of Myc-TDP-43 compared with TDP-43-V5-WT is likely because its plasmid backbone does not replicate and is diluted out of cells during the course of antibiotic selection which spans a couple of weeks

Article Snippet: 99-PUR-RPS-EGFP includes full-length L1-RP with the EGFP reporter cassette in its 3' UTR cloned in a modified version of pCEP4 vector (Invitrogen) lacking a cytomegalovirus (CMV) promoter.

Techniques: Expressing, Cell Culture, Infection, Flow Cytometry, Transfection, Plasmid Preparation, Construct, Western Blot, FLAG-tag, Molecular Weight, Gel Purification, Staining, Selection