Journal: Cell reports

Article Title: Pharmacological inhibition of neddylation impairs long interspersed element 1 retrotransposition.

doi: 10.1016/j.celrep.2024.113749

Figure Lengend Snippet: Figure 5. Dominant-negative human Cullins suppress the capacity of L1 ORF2p reverse transcriptase to reduce L1 retrotransposition (A) Empty (pcDNA3.1[+]) and dominant-negative Cullin (Cul-DN) vectors were co-transfected with L1RP EGFP into HEK293T cells. JM111 was used as a negative control. The bar graph represents the EGFP-positive cell fraction. Data are presented as mean ± SD. **p < 0.01; n.s., not significant. (B) Protein analysis of Cullin 1–5 in HEK293T cells transfected with siRNAs was conducted using western blotting with anti-Cullin 1–5 antibodies. (C) HEK293T cells pre-transfected with siRNAs were transfected with L1RP EGFP plasmids. GFP (+) cells were detected using flow cytometry. The bar graph indicates the mean ± SD of the fraction of GFP-positive cells. **p < 0.01; n.s., not significant. (D) pc-L1-1FH plasmids were transfected into HEK293T cells, and protein samples were prepared and immunoblotted using an anti-HA antibody. (E) Empty vector (pcDNA3.1[+]), Cul1-DN, Cul3-DN, Cul4A-DN, or Cul4B-DN plasmids were co-transfected with pEGFP-N1-ORF1-EGFP. Images were obtained 48 h post transfection. Scale bar, 20 mm. (F) LEAP assay was performed as described in the STAR Methods section. Products from the MuLV RT and LEAP assays were subjected to PCR amplification and were visualized using gel electrophoresis. The amount of L1 ORF1p in the L1 RNP was determined by immunoblotting using an anti-HA antibody. (G) real-time qPCR analysis of MuLV RT and LEAP products. Relative cDNA levels were set to 1.0. Data are presented as mean ± SD. **p < 0.01; n.s., not significant. L1, long interspersed element 1; real-time qPCR, quantitative real-time polymerase chain reaction; RT, reverse transcriptase; LEAP, L1 element amplification protocol; RNP, ribonucleoprotein. Experiments in this figure were reproduced at least three times, and a representative experiment is shown. The bar chart was based on three replicates within one experiment.

Article Snippet: Moreover, L1-neo-TET (Addgene, 51284), dominant negative Cullin 2 (Addgene, 41912), Cullin 4A (Addgene, 41914), Cullin 4B (Addgene, 41915), and Cullin 5 (Addgene, 41916) plasmids were purchased from Addgene.

Techniques: Dominant Negative Mutation, Reverse Transcription, Transfection, Negative Control, Western Blot, Cytometry, Plasmid Preparation, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction