anti nrbp1 2 antibody  (Proteintech)

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) The NRBP1/2 antibody used for MS interactome analysis recognizes both NRBP1 and NRBP2. (B) Both NRBP1 and NRBP2 are associated with multiple known L1 interactors or regulators. HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Myc-Flag-NRBP1 or Myc-Flag-NRBP2, co-precipitated endogenous proteins were detected by using respective antibodies. Myc-Flag-NRBP1 and Myc-Flag-NRBP2 were detected with Myc antibody.

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Scatter plots showing proteins significantly enriched (fold change ≥ 5, p -value < 0.05) in NRBP1/2 IP vs. IgG in MCF-7 cells. NRBP1 and NRBP2 are shown in red. Previously known NRBP1 interactors are labelled in blue. ORF1 is marked in orange. (B) Endogenous ORF1 is co-immunoprecipitated with NRBP1 and/or NRBP2. An antibody recognizing both NRBP1 and NRBP2 was used to pull down endogenous NRBP1 and NRBP2 in MCF-7 cells. Co-precipitated ORF1 was detected by using ORF1 antibody. (C) Endogenous ORF1 is co-immunoprecipitated with both NRBP1 and NRBP2. MCF-7 cells were transfected with either Myc-Flag-tagged NRBP1 or Myc-Flag-tagged NRBP2. The cell lysates were incubated with Flag antibody and the immunoprecipitated proteins were detected by Western blot with the indicated antibodies. (D) NRBP1 and NRBP2 are co-immunoprecipitated with ORF1. Flag antibody was used to pull down ORF1-Flag, and Myc antibody was used to detect co-immunoprecipitated Myc-NRBP1 and Myc-NRBP2 in HeLa cells. (E-L) NRBP1 activates and NRBP2 inhibits L1 retrotransposition. One representative L1 retrotransposition activity is shown in (E) , (G) , (I) and (K) . Quantitation of L1 retrotransposition activity is shown in the top panels of (F) , (H) , (J) and (L) . Western blots indicating successful overexpression or knockdown are shown in the bottom panels of (F) , (H) , (J) and (L) . N = 4 biological replicates for (E) and (F) . N = 3 biological replicates for (G) and (H) . N = 7 biological replicates for (I) and (J) . N = 3 biological replicates for (K) and (L) . See also and Table S1.

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunoprecipitation, Transfection, Incubation, Western Blot, Activity Assay, Quantitation Assay, Over Expression

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) MCF-7 cells show higher ORF1 protein level than HeLa cells. (B) Western blot assesses efficiency of NRBP1 knockdown and ORF1 IP for the experiments in . (C) Induction of ORF1-Flag enriched foci by NRBP2 overexpression is independent of G3BP1. HA-NRBP2, ORF1-Flag and G3BP1 were stained by HA, Flag and G3BP1 antibodies, respectively. Scale bar 10 µm. (D) Inhibition of L1 retrotransposition by NRBP2 is independent of G3BP1. Quantification of the colony assay is shown in the top panel and one representative Western blot result is shown in the bottom panel. N = 4. (E) One representative picture of the colony assay depicted in (D) . (F) Western blot assesses efficiency of HA-NRBP2 transfection and ORF1 IP for the experiments in .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Western Blot, Over Expression, Staining, Inhibition, Colony Assay, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown results in an enrichment of ORF1 in cytoplasmic foci in MCF-7 cells. The yellow arrows point to the ORF1 foci. Shown are immunofluorescence staining of endogenous ORF1 and G3BP1 proteins with their respective antibodies. Scale bar 10 µm. (B) NRBP1 knockdown impairs colocalization of ORF1 and L1 mRNA. Shown are immunofluorescence staining of endogenous ORF1 protein with ORF1 antibody (green) and L1 mRNAs detected via smFISH with L1 probes (violet). Nucleus was stained with Hoechst 33342 (blue). The arrows in the top panel point to co-localized ORF1 and L1 mRNA, the arrows in the bottom panel point to the ORF1-enriched puncta without an enrichment of L1 mRNA. Scale bar 10 µm. (C) ORF1 RIP-qPCR to quantitate ORF1-associated L1 mRNA. Three primer pairs targeting to L1 5′ UTR, ORF1 and ORF2 were used for qPCR. N = 3 biological replicates. (D) HA-NRBP2 overexpression results in an enrichment of ORF1-Flag in cytoplasmic puncta in HeLa cells. The yellow arrows point to colocalized ORF1 and G3BP1 foci. Scale bar 10 µm. (E) NRBP2 overexpression prevents the interaction between ORF1 protein and L1 mRNA. RIP-qPCR was carried out to quantitate Flag-tagged ORF1 co-immunoprecipitated L1 mRNA levels with indicated primer pairs. N = 3 biological replicates. See also .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Over Expression, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Overexpressing NRBP1 or NRBP2 does not interfere with binding of their ortholog counterpart to ORF1. Co-IP was performed in HeLa cells by using Flag antibody. (B) NRBP1 knockdown abolishes inhibitory effect of NRBP2 on L1 retrotransposition. Shown is a representative picture of the colony assay depicted in . (C) NRBP2 knockdown does not significantly increase mRNA level of NRBP1. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates. (D) NRBP1 knockdown increases protein level of NRBP2. Shown is quantification of four replicates in . (E) NRBP1 knockdown increases mRNA level of NRBP2. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates.

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown abolishes the inhibitory effect of NRBP2 on L1 in HeLa cells. Shown is quantitation of L1 retrotransposition activity. N = 4 biological replicates. (B) Overexpression of NRBP2-Myc reduces endogenous NRBP1 protein level. Shown is one representative Western blot using the samples from (A) to detect endogenous NRBP1 and transfected NRBP2-Myc. (C and D) NRBP1 and NRBP2 negatively affect the protein levels of each other in HeLa cells. Endogenous NRBP1 and NRBP2 were detected using NRBP1/2 antibody. Shown is one representative Western blot result (C) . Quantification of the protein levels was shown in (D) . N = 4 biological replicates. (E) Overexpressing NRBP2-Myc results in an enrichment of HA-NRBP1 to the peripheral region of HeLa cells. Yellow arrows point to cells co-expressing HA-NRBP1 and NRBP2-Myc. White arrow indicates a cell only expressing NRBP1. HA-NRBP1 and NRBP2-Myc were detected using HA and Myc antibodies. Scale bar 10 µm. See also .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Quantitation Assay, Activity Assay, Over Expression, Western Blot, Transfection, Expressing

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Diagram illustrations of domains / motifs in human NRBP1 and NRBP2 proteins. (B) NRBP1 structure prediction by AlphaFold (AF-Q9UHY1-F1) reveals a potential unstructured N-terminal LCR. (C) A simple schematic diagram of NRBP1, NRBP2 and their mutants for the LCR-swapping experiment. (D) A representative Western blot to show expression of NRBP1, NRBP2 and their mutants in (C) and (E) . (E) LCR-swapping does not interfere with the regulatory roles of NRBP1 and NRBP2 in L1 mobility. Shown is one representative picture of the colony assay to examine L1 mobility. (F) NRB and dimerization region of NRBP2 are essential for its inhibitory role on L1. Shown is one representative picture of the colony assay depicted in . (G) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is one representative picture of the colony assay depicted in . (H) Both C- and N-terminal halves of NRBP2 interact with ORF1 in HEK293T cells. (I) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is one representative picture of the colony assay depicted in .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Schematic diagrams of NRBP2 and the NRBP2 mutants used in this study. NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (B) The NRB and dimerization region of NRBP2 are required for NRBP2 to inhibit L1 retrotransposition. Quantification of L1 activity is shown in the top panel. N = 5 biological replicates. One representative Western blot showing expression of the respective NRBP2 mutant variants is shown in the bottom panel. (C) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is quantification of L1 activity upon expression of the respective NRBP2 variants. N = 3 biological replicates. (D) Schematic diagram of NRBP1 and the NRBP1 halves used in this study. LCR: low complexity region; NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (E) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is quantification of L1 activity upon expression of the respective NRBP1 variants. For NRBP1, N = 5 biological replicates. For NRBP1-N and NRBP1-C, N = 3 biological replicates. (F) The C-terminal half of NRBP2 promotes enrichment of NRBP1 to the peripheral region of HeLa cells. White arrows point to NRBP1 without NRBP2 transfection. Yellow arrows indicate cells with enrichment of NRBP1 to the peripheral region upon NRBP2 overexpression. Scale bar 10 µm. (G) The C-terminal half of NRBP1 promotes enrichment of full-length NRBP1 to the peripheral region of HeLa cells. Scale bar 10 µm. (H) Overexpressing NRBP2 and C-terminal half of NRBP1 or NRBP2 reduces endogenous NRBP1 protein level in HeLa cells. Shown is one representative Western blot to detect the respective NRBP1 or NRBP2 variants and endogenous NRBP1. N = 3 biological replicates. See also , and .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, Mutagenesis, Transfection, Over Expression

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Proteasome inhibitor MG132 abolishes capability of NRBP2 and the C-terminal halves of NRBP1 and NRBP2 to reduce protein level of NRBP1. Shown is quantification of relative protein levels of NRBP1 in HeLa cells. GAPDH was used for normalization. N = 3 biological replicates. (B) Proteasome inhibitor MG132 prevents enrichment of NRBP1 to the peripheral region of HeLa cells upon overexpressing the C-terminal half of NRBP1 or NRBP2. Shown are representative confocal images of immunofluorescence stained cells. Scale bar 10 µm. (C) NRB motif in NRBP2 is essential to reduce protein level of NRBP1 in HeLa cells. The upper panel shows the quantification of the relative NRBP1 protein level normalized by GAPDH. Bottom panel is one representative Western blot used for quantification. N = 3 biological replicates. (D) NRB motifs in C-terminal half of NRBP1 are essential to inhibit L1 and to reduce the protein level of NRBP1. Quantification of L1 activity in HeLa cells is shown in the top panel. N = 3 biological replicates. One representative Western blot showing expression of the respective proteins is shown in the bottom panel. (E) The C-terminal half of NRBP1 interacts with the full-length NRBP1 in an NRB-dependent manner. (F) The C-terminal half of NRBP2 interacts with the full-length NRBP1 in an NRB-dependent manner. (G) Lack of NRB and dimerization region does not prevent interaction between NRBP2 and NRBP1. (H) Both the N- and C-terminal halves of NRBP2 interact with NRBP1. For E-H , HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Flag-NRBP1. The co-precipitated proteins were detected by using HA or Myc antibody. See also and S7.

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay, Expressing, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 reduce protein level of full-length NRBP1 in HeLa cells and this could be blocked with MG132 treatment. Shown is one representative Western blot result used for the quantification in the main . (B) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 promote NRBP1 decay independently of ELOB and ELOC. In line with prior findings, reducing either ELOB or ELOC resulted in a decrease in the protein levels of the other. , Shown is one representative Western blot result used for the quantification in (C) . (C) Quantification of three independent Western blot experiments from (B) . (D) The NRB motifs in the C-terminal half of NRBP1 are essential to inhibit L1. Shown is one representative picture of the colony assay depicted in the main .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Western Blot, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A and B) Pie charts illustrating the numbers of genes displaying significantly altered expression levels (fold change ≥ 1.5 and FDR < 0.05) following the knockdown of either NRBP2 (A) or NRBP1 (B) in HeLa cells. (C) Venn Diagram showing overlapping genes upregulated by NRBP2 knockdown in HeLa cells and genes activated upon L1 overexpression in RPE cells. p < 2.081e-25. The p-value was calculated using http://nemates.org/MA/progs/overlap_stats.html . to determine the statistical significance of the overlap between the two groups of genes. (D) GO term analysis for the overlapping genes in (C) with the online software DAVID. Pathways exhibiting an enrichment with -log 10 p-value >2 are presented. (E) qRT-PCR to confirm activation of selected innate immunity genes upon NRBP2 knockdown in HeLa cells. N = 3 biological replicates. (F) mRNA levels of NRBP1 and NRBP2 in samples obtained from both healthy individuals and RA patients. Data is taken from Autoimmune Diseases Explorer ( https://adex.genyo.es ). See also Table S2.

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Expressing, Over Expression, Software, Quantitative RT-PCR, Activation Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Maximum likelihood phylogeny of the IQ-TREE analysis comprising 2,065 quality-filtered NRBP proteins belonging to 663 species and including 417 amino acid positions. Orthologues of human NRBP2 are recovered as a monophyletic group with perfect bootstrap support (blue). All NRBP2 belong to the Euteleostomi. Its sister clade contains all orthologues of human NRBP1 derived from vertebrate taxa (orange) and has perfect bootstrap support as well. The NRBP sequence of the sponge Amphimedon queenslandica was chosen as outgroup taxon. Branch lengths are amino acid substitutions per site. (B) The distance from leaf to root was determined in (A) for all NRBP proteins. The NRBP2 clade accumulated considerably more amino acid substitutions than the NRBP1 clade and the average invertebrate NRBP protein. A consequence of this is that all invertebrate NRBP proteins are more similar to human NRBP1 than they are to human NRBP2. (C) Scatter plot of BlastP bit scores resulting from 462 comparisons of invertebrate NRBP either to human NRBP1 or to human NRBP2. For all value pairs, the bit score for the NRBP1 comparison is higher than that of the respective comparison to NRBP2. (D) Plot and paired t-test for the values described in (C) results in a p-value of 1.5 × 10 -202 . This indicates that human NRBP1 is more similar to invertebrate NRBP than human NRBP2 is. See also .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Comparison

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) We mapped 2,065 homologs of human NRBP1/2 to a phylogenetic tree of eukaryotes. Clades drawn with red lines indicate the presence of NRBP , whereas clades drawn with black lines indicate the absence of NRBP . NRBP is present in the early metazoan branches Porifera and Placozoa, but it is absent from all non-metazoan eukaryotes. The figure is a modified image from . The original image is licensed under the Creative Commons Attribution 2.5 Generic license ( https://creativecommons.org/licenses/by/2.5/deed.en ). (B) We mapped 669 candidate orthologues of human NRBP2 to a phylogenetic tree of vertebrates. Clades drawn with red lines indicate the presence of NRBP2 , whereas clades drawn with black lines indicate the absence of NRBP2 . All NRBP2 map either to the Sarcopterygii or to the Actinopterygii, indicating an evolutionary origin of NRBP2 either in the Eutelestomi, or earlier in vertebrate evolution. All presented clades (black and red lines) have candidate orthologues of NRBP1 . Remarkably, clades with NRBP2 have a ‘Type II’ L1 ORF1, which is characterised by ‘transposase 22’, whereas the clades lacking NRBP2 also lack the transposase 22. For details, please refer to the discussion section. The image is modified from https://en.wikipedia.org/wiki/Actinopterygii .

Article Snippet: HeLa cells were transfected with a final concentration of either 20 nM siRNA (for NRBP1 and NRBP2 knockdown) or 10 nM siRNA (for ELOB and ELOC knockdown) in two consecutive days using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Modification

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) The NRBP1/2 antibody used for MS interactome analysis recognizes both NRBP1 and NRBP2. (B) Both NRBP1 and NRBP2 are associated with multiple known L1 interactors or regulators. HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Myc-Flag-NRBP1 or Myc-Flag-NRBP2, co-precipitated endogenous proteins were detected by using respective antibodies. Myc-Flag-NRBP1 and Myc-Flag-NRBP2 were detected with Myc antibody.

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Scatter plots showing proteins significantly enriched (fold change ≥ 5, p -value < 0.05) in NRBP1/2 IP vs. IgG in MCF-7 cells. NRBP1 and NRBP2 are shown in red. Previously known NRBP1 interactors are labelled in blue. ORF1 is marked in orange. (B) Endogenous ORF1 is co-immunoprecipitated with NRBP1 and/or NRBP2. An antibody recognizing both NRBP1 and NRBP2 was used to pull down endogenous NRBP1 and NRBP2 in MCF-7 cells. Co-precipitated ORF1 was detected by using ORF1 antibody. (C) Endogenous ORF1 is co-immunoprecipitated with both NRBP1 and NRBP2. MCF-7 cells were transfected with either Myc-Flag-tagged NRBP1 or Myc-Flag-tagged NRBP2. The cell lysates were incubated with Flag antibody and the immunoprecipitated proteins were detected by Western blot with the indicated antibodies. (D) NRBP1 and NRBP2 are co-immunoprecipitated with ORF1. Flag antibody was used to pull down ORF1-Flag, and Myc antibody was used to detect co-immunoprecipitated Myc-NRBP1 and Myc-NRBP2 in HeLa cells. (E-L) NRBP1 activates and NRBP2 inhibits L1 retrotransposition. One representative L1 retrotransposition activity is shown in (E) , (G) , (I) and (K) . Quantitation of L1 retrotransposition activity is shown in the top panels of (F) , (H) , (J) and (L) . Western blots indicating successful overexpression or knockdown are shown in the bottom panels of (F) , (H) , (J) and (L) . N = 4 biological replicates for (E) and (F) . N = 3 biological replicates for (G) and (H) . N = 7 biological replicates for (I) and (J) . N = 3 biological replicates for (K) and (L) . See also and Table S1.

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Immunoprecipitation, Transfection, Incubation, Western Blot, Activity Assay, Quantitation Assay, Over Expression, Knockdown

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) MCF-7 cells show higher ORF1 protein level than HeLa cells. (B) Western blot assesses efficiency of NRBP1 knockdown and ORF1 IP for the experiments in . (C) Induction of ORF1-Flag enriched foci by NRBP2 overexpression is independent of G3BP1. HA-NRBP2, ORF1-Flag and G3BP1 were stained by HA, Flag and G3BP1 antibodies, respectively. Scale bar 10 µm. (D) Inhibition of L1 retrotransposition by NRBP2 is independent of G3BP1. Quantification of the colony assay is shown in the top panel and one representative Western blot result is shown in the bottom panel. N = 4. (E) One representative picture of the colony assay depicted in (D) . (F) Western blot assesses efficiency of HA-NRBP2 transfection and ORF1 IP for the experiments in .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Western Blot, Knockdown, Over Expression, Staining, Inhibition, Colony Assay, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown results in an enrichment of ORF1 in cytoplasmic foci in MCF-7 cells. The yellow arrows point to the ORF1 foci. Shown are immunofluorescence staining of endogenous ORF1 and G3BP1 proteins with their respective antibodies. Scale bar 10 µm. (B) NRBP1 knockdown impairs colocalization of ORF1 and L1 mRNA. Shown are immunofluorescence staining of endogenous ORF1 protein with ORF1 antibody (green) and L1 mRNAs detected via smFISH with L1 probes (violet). Nucleus was stained with Hoechst 33342 (blue). The arrows in the top panel point to co-localized ORF1 and L1 mRNA, the arrows in the bottom panel point to the ORF1-enriched puncta without an enrichment of L1 mRNA. Scale bar 10 µm. (C) ORF1 RIP-qPCR to quantitate ORF1-associated L1 mRNA. Three primer pairs targeting to L1 5′ UTR, ORF1 and ORF2 were used for qPCR. N = 3 biological replicates. (D) HA-NRBP2 overexpression results in an enrichment of ORF1-Flag in cytoplasmic puncta in HeLa cells. The yellow arrows point to colocalized ORF1 and G3BP1 foci. Scale bar 10 µm. (E) NRBP2 overexpression prevents the interaction between ORF1 protein and L1 mRNA. RIP-qPCR was carried out to quantitate Flag-tagged ORF1 co-immunoprecipitated L1 mRNA levels with indicated primer pairs. N = 3 biological replicates. See also .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Knockdown, Immunofluorescence, Staining, Over Expression, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Overexpressing NRBP1 or NRBP2 does not interfere with binding of their ortholog counterpart to ORF1. Co-IP was performed in HeLa cells by using Flag antibody. (B) NRBP1 knockdown abolishes inhibitory effect of NRBP2 on L1 retrotransposition. Shown is a representative picture of the colony assay depicted in . (C) NRBP2 knockdown does not significantly increase mRNA level of NRBP1. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates. (D) NRBP1 knockdown increases protein level of NRBP2. Shown is quantification of four replicates in . (E) NRBP1 knockdown increases mRNA level of NRBP2. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates.

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown abolishes the inhibitory effect of NRBP2 on L1 in HeLa cells. Shown is quantitation of L1 retrotransposition activity. N = 4 biological replicates. (B) Overexpression of NRBP2-Myc reduces endogenous NRBP1 protein level. Shown is one representative Western blot using the samples from (A) to detect endogenous NRBP1 and transfected NRBP2-Myc. (C and D) NRBP1 and NRBP2 negatively affect the protein levels of each other in HeLa cells. Endogenous NRBP1 and NRBP2 were detected using NRBP1/2 antibody. Shown is one representative Western blot result (C) . Quantification of the protein levels was shown in (D) . N = 4 biological replicates. (E) Overexpressing NRBP2-Myc results in an enrichment of HA-NRBP1 to the peripheral region of HeLa cells. Yellow arrows point to cells co-expressing HA-NRBP1 and NRBP2-Myc. White arrow indicates a cell only expressing NRBP1. HA-NRBP1 and NRBP2-Myc were detected using HA and Myc antibodies. Scale bar 10 µm. See also .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Knockdown, Quantitation Assay, Activity Assay, Over Expression, Western Blot, Transfection, Expressing

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Diagram illustrations of domains / motifs in human NRBP1 and NRBP2 proteins. (B) NRBP1 structure prediction by AlphaFold (AF-Q9UHY1-F1) reveals a potential unstructured N-terminal LCR. (C) A simple schematic diagram of NRBP1, NRBP2 and their mutants for the LCR-swapping experiment. (D) A representative Western blot to show expression of NRBP1, NRBP2 and their mutants in (C) and (E) . (E) LCR-swapping does not interfere with the regulatory roles of NRBP1 and NRBP2 in L1 mobility. Shown is one representative picture of the colony assay to examine L1 mobility. (F) NRB and dimerization region of NRBP2 are essential for its inhibitory role on L1. Shown is one representative picture of the colony assay depicted in . (G) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is one representative picture of the colony assay depicted in . (H) Both C- and N-terminal halves of NRBP2 interact with ORF1 in HEK293T cells. (I) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is one representative picture of the colony assay depicted in .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Western Blot, Expressing, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Schematic diagrams of NRBP2 and the NRBP2 mutants used in this study. NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (B) The NRB and dimerization region of NRBP2 are required for NRBP2 to inhibit L1 retrotransposition. Quantification of L1 activity is shown in the top panel. N = 5 biological replicates. One representative Western blot showing expression of the respective NRBP2 mutant variants is shown in the bottom panel. (C) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is quantification of L1 activity upon expression of the respective NRBP2 variants. N = 3 biological replicates. (D) Schematic diagram of NRBP1 and the NRBP1 halves used in this study. LCR: low complexity region; NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (E) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is quantification of L1 activity upon expression of the respective NRBP1 variants. For NRBP1, N = 5 biological replicates. For NRBP1-N and NRBP1-C, N = 3 biological replicates. (F) The C-terminal half of NRBP2 promotes enrichment of NRBP1 to the peripheral region of HeLa cells. White arrows point to NRBP1 without NRBP2 transfection. Yellow arrows indicate cells with enrichment of NRBP1 to the peripheral region upon NRBP2 overexpression. Scale bar 10 µm. (G) The C-terminal half of NRBP1 promotes enrichment of full-length NRBP1 to the peripheral region of HeLa cells. Scale bar 10 µm. (H) Overexpressing NRBP2 and C-terminal half of NRBP1 or NRBP2 reduces endogenous NRBP1 protein level in HeLa cells. Shown is one representative Western blot to detect the respective NRBP1 or NRBP2 variants and endogenous NRBP1. N = 3 biological replicates. See also , and .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, Mutagenesis, Transfection, Over Expression

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Proteasome inhibitor MG132 abolishes capability of NRBP2 and the C-terminal halves of NRBP1 and NRBP2 to reduce protein level of NRBP1. Shown is quantification of relative protein levels of NRBP1 in HeLa cells. GAPDH was used for normalization. N = 3 biological replicates. (B) Proteasome inhibitor MG132 prevents enrichment of NRBP1 to the peripheral region of HeLa cells upon overexpressing the C-terminal half of NRBP1 or NRBP2. Shown are representative confocal images of immunofluorescence stained cells. Scale bar 10 µm. (C) NRB motif in NRBP2 is essential to reduce protein level of NRBP1 in HeLa cells. The upper panel shows the quantification of the relative NRBP1 protein level normalized by GAPDH. Bottom panel is one representative Western blot used for quantification. N = 3 biological replicates. (D) NRB motifs in C-terminal half of NRBP1 are essential to inhibit L1 and to reduce the protein level of NRBP1. Quantification of L1 activity in HeLa cells is shown in the top panel. N = 3 biological replicates. One representative Western blot showing expression of the respective proteins is shown in the bottom panel. (E) The C-terminal half of NRBP1 interacts with the full-length NRBP1 in an NRB-dependent manner. (F) The C-terminal half of NRBP2 interacts with the full-length NRBP1 in an NRB-dependent manner. (G) Lack of NRB and dimerization region does not prevent interaction between NRBP2 and NRBP1. (H) Both the N- and C-terminal halves of NRBP2 interact with NRBP1. For E-H , HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Flag-NRBP1. The co-precipitated proteins were detected by using HA or Myc antibody. See also and S7.

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay, Expressing, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 reduce protein level of full-length NRBP1 in HeLa cells and this could be blocked with MG132 treatment. Shown is one representative Western blot result used for the quantification in the main . (B) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 promote NRBP1 decay independently of ELOB and ELOC. In line with prior findings, reducing either ELOB or ELOC resulted in a decrease in the protein levels of the other. , Shown is one representative Western blot result used for the quantification in (C) . (C) Quantification of three independent Western blot experiments from (B) . (D) The NRB motifs in the C-terminal half of NRBP1 are essential to inhibit L1. Shown is one representative picture of the colony assay depicted in the main .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Western Blot, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A and B) Pie charts illustrating the numbers of genes displaying significantly altered expression levels (fold change ≥ 1.5 and FDR < 0.05) following the knockdown of either NRBP2 (A) or NRBP1 (B) in HeLa cells. (C) Venn Diagram showing overlapping genes upregulated by NRBP2 knockdown in HeLa cells and genes activated upon L1 overexpression in RPE cells. p < 2.081e-25. The p-value was calculated using http://nemates.org/MA/progs/overlap_stats.html . to determine the statistical significance of the overlap between the two groups of genes. (D) GO term analysis for the overlapping genes in (C) with the online software DAVID. Pathways exhibiting an enrichment with -log 10 p-value >2 are presented. (E) qRT-PCR to confirm activation of selected innate immunity genes upon NRBP2 knockdown in HeLa cells. N = 3 biological replicates. (F) mRNA levels of NRBP1 and NRBP2 in samples obtained from both healthy individuals and RA patients. Data is taken from Autoimmune Diseases Explorer ( https://adex.genyo.es ). See also Table S2.

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Expressing, Knockdown, Over Expression, Software, Quantitative RT-PCR, Activation Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Maximum likelihood phylogeny of the IQ-TREE analysis comprising 2,065 quality-filtered NRBP proteins belonging to 663 species and including 417 amino acid positions. Orthologues of human NRBP2 are recovered as a monophyletic group with perfect bootstrap support (blue). All NRBP2 belong to the Euteleostomi. Its sister clade contains all orthologues of human NRBP1 derived from vertebrate taxa (orange) and has perfect bootstrap support as well. The NRBP sequence of the sponge Amphimedon queenslandica was chosen as outgroup taxon. Branch lengths are amino acid substitutions per site. (B) The distance from leaf to root was determined in (A) for all NRBP proteins. The NRBP2 clade accumulated considerably more amino acid substitutions than the NRBP1 clade and the average invertebrate NRBP protein. A consequence of this is that all invertebrate NRBP proteins are more similar to human NRBP1 than they are to human NRBP2. (C) Scatter plot of BlastP bit scores resulting from 462 comparisons of invertebrate NRBP either to human NRBP1 or to human NRBP2. For all value pairs, the bit score for the NRBP1 comparison is higher than that of the respective comparison to NRBP2. (D) Plot and paired t-test for the values described in (C) results in a p-value of 1.5 × 10 -202 . This indicates that human NRBP1 is more similar to invertebrate NRBP than human NRBP2 is. See also .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Derivative Assay, Sequencing, Comparison

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) We mapped 2,065 homologs of human NRBP1/2 to a phylogenetic tree of eukaryotes. Clades drawn with red lines indicate the presence of NRBP , whereas clades drawn with black lines indicate the absence of NRBP . NRBP is present in the early metazoan branches Porifera and Placozoa, but it is absent from all non-metazoan eukaryotes. The figure is a modified image from . The original image is licensed under the Creative Commons Attribution 2.5 Generic license ( https://creativecommons.org/licenses/by/2.5/deed.en ). (B) We mapped 669 candidate orthologues of human NRBP2 to a phylogenetic tree of vertebrates. Clades drawn with red lines indicate the presence of NRBP2 , whereas clades drawn with black lines indicate the absence of NRBP2 . All NRBP2 map either to the Sarcopterygii or to the Actinopterygii, indicating an evolutionary origin of NRBP2 either in the Eutelestomi, or earlier in vertebrate evolution. All presented clades (black and red lines) have candidate orthologues of NRBP1 . Remarkably, clades with NRBP2 have a ‘Type II’ L1 ORF1, which is characterised by ‘transposase 22’, whereas the clades lacking NRBP2 also lack the transposase 22. For details, please refer to the discussion section. The image is modified from https://en.wikipedia.org/wiki/Actinopterygii .

Article Snippet: ON-TARGET plus SMARTpool siRNAs directed against NRBP1(L-005356-00), NRBP2 (L-005340-02), ELOB (L-012376-00), ELOC (L-010541-00) and Non-targeting control (siControl) siRNA (D-001810-10) were purchased from Dharmacon.

Techniques: Modification

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) The NRBP1/2 antibody used for MS interactome analysis recognizes both NRBP1 and NRBP2. (B) Both NRBP1 and NRBP2 are associated with multiple known L1 interactors or regulators. HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Myc-Flag-NRBP1 or Myc-Flag-NRBP2, co-precipitated endogenous proteins were detected by using respective antibodies. Myc-Flag-NRBP1 and Myc-Flag-NRBP2 were detected with Myc antibody.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Scatter plots showing proteins significantly enriched (fold change ≥ 5, p -value < 0.05) in NRBP1/2 IP vs. IgG in MCF-7 cells. NRBP1 and NRBP2 are shown in red. Previously known NRBP1 interactors are labelled in blue. ORF1 is marked in orange. (B) Endogenous ORF1 is co-immunoprecipitated with NRBP1 and/or NRBP2. An antibody recognizing both NRBP1 and NRBP2 was used to pull down endogenous NRBP1 and NRBP2 in MCF-7 cells. Co-precipitated ORF1 was detected by using ORF1 antibody. (C) Endogenous ORF1 is co-immunoprecipitated with both NRBP1 and NRBP2. MCF-7 cells were transfected with either Myc-Flag-tagged NRBP1 or Myc-Flag-tagged NRBP2. The cell lysates were incubated with Flag antibody and the immunoprecipitated proteins were detected by Western blot with the indicated antibodies. (D) NRBP1 and NRBP2 are co-immunoprecipitated with ORF1. Flag antibody was used to pull down ORF1-Flag, and Myc antibody was used to detect co-immunoprecipitated Myc-NRBP1 and Myc-NRBP2 in HeLa cells. (E-L) NRBP1 activates and NRBP2 inhibits L1 retrotransposition. One representative L1 retrotransposition activity is shown in (E) , (G) , (I) and (K) . Quantitation of L1 retrotransposition activity is shown in the top panels of (F) , (H) , (J) and (L) . Western blots indicating successful overexpression or knockdown are shown in the bottom panels of (F) , (H) , (J) and (L) . N = 4 biological replicates for (E) and (F) . N = 3 biological replicates for (G) and (H) . N = 7 biological replicates for (I) and (J) . N = 3 biological replicates for (K) and (L) . See also and Table S1.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Immunoprecipitation, Transfection, Incubation, Western Blot, Activity Assay, Quantitation Assay, Over Expression, Knockdown

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) MCF-7 cells show higher ORF1 protein level than HeLa cells. (B) Western blot assesses efficiency of NRBP1 knockdown and ORF1 IP for the experiments in . (C) Induction of ORF1-Flag enriched foci by NRBP2 overexpression is independent of G3BP1. HA-NRBP2, ORF1-Flag and G3BP1 were stained by HA, Flag and G3BP1 antibodies, respectively. Scale bar 10 µm. (D) Inhibition of L1 retrotransposition by NRBP2 is independent of G3BP1. Quantification of the colony assay is shown in the top panel and one representative Western blot result is shown in the bottom panel. N = 4. (E) One representative picture of the colony assay depicted in (D) . (F) Western blot assesses efficiency of HA-NRBP2 transfection and ORF1 IP for the experiments in .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Western Blot, Knockdown, Over Expression, Staining, Inhibition, Colony Assay, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown results in an enrichment of ORF1 in cytoplasmic foci in MCF-7 cells. The yellow arrows point to the ORF1 foci. Shown are immunofluorescence staining of endogenous ORF1 and G3BP1 proteins with their respective antibodies. Scale bar 10 µm. (B) NRBP1 knockdown impairs colocalization of ORF1 and L1 mRNA. Shown are immunofluorescence staining of endogenous ORF1 protein with ORF1 antibody (green) and L1 mRNAs detected via smFISH with L1 probes (violet). Nucleus was stained with Hoechst 33342 (blue). The arrows in the top panel point to co-localized ORF1 and L1 mRNA, the arrows in the bottom panel point to the ORF1-enriched puncta without an enrichment of L1 mRNA. Scale bar 10 µm. (C) ORF1 RIP-qPCR to quantitate ORF1-associated L1 mRNA. Three primer pairs targeting to L1 5′ UTR, ORF1 and ORF2 were used for qPCR. N = 3 biological replicates. (D) HA-NRBP2 overexpression results in an enrichment of ORF1-Flag in cytoplasmic puncta in HeLa cells. The yellow arrows point to colocalized ORF1 and G3BP1 foci. Scale bar 10 µm. (E) NRBP2 overexpression prevents the interaction between ORF1 protein and L1 mRNA. RIP-qPCR was carried out to quantitate Flag-tagged ORF1 co-immunoprecipitated L1 mRNA levels with indicated primer pairs. N = 3 biological replicates. See also .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Knockdown, Immunofluorescence, Staining, Over Expression, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Overexpressing NRBP1 or NRBP2 does not interfere with binding of their ortholog counterpart to ORF1. Co-IP was performed in HeLa cells by using Flag antibody. (B) NRBP1 knockdown abolishes inhibitory effect of NRBP2 on L1 retrotransposition. Shown is a representative picture of the colony assay depicted in . (C) NRBP2 knockdown does not significantly increase mRNA level of NRBP1. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates. (D) NRBP1 knockdown increases protein level of NRBP2. Shown is quantification of four replicates in . (E) NRBP1 knockdown increases mRNA level of NRBP2. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown abolishes the inhibitory effect of NRBP2 on L1 in HeLa cells. Shown is quantitation of L1 retrotransposition activity. N = 4 biological replicates. (B) Overexpression of NRBP2-Myc reduces endogenous NRBP1 protein level. Shown is one representative Western blot using the samples from (A) to detect endogenous NRBP1 and transfected NRBP2-Myc. (C and D) NRBP1 and NRBP2 negatively affect the protein levels of each other in HeLa cells. Endogenous NRBP1 and NRBP2 were detected using NRBP1/2 antibody. Shown is one representative Western blot result (C) . Quantification of the protein levels was shown in (D) . N = 4 biological replicates. (E) Overexpressing NRBP2-Myc results in an enrichment of HA-NRBP1 to the peripheral region of HeLa cells. Yellow arrows point to cells co-expressing HA-NRBP1 and NRBP2-Myc. White arrow indicates a cell only expressing NRBP1. HA-NRBP1 and NRBP2-Myc were detected using HA and Myc antibodies. Scale bar 10 µm. See also .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Knockdown, Quantitation Assay, Activity Assay, Over Expression, Western Blot, Transfection, Expressing

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Diagram illustrations of domains / motifs in human NRBP1 and NRBP2 proteins. (B) NRBP1 structure prediction by AlphaFold (AF-Q9UHY1-F1) reveals a potential unstructured N-terminal LCR. (C) A simple schematic diagram of NRBP1, NRBP2 and their mutants for the LCR-swapping experiment. (D) A representative Western blot to show expression of NRBP1, NRBP2 and their mutants in (C) and (E) . (E) LCR-swapping does not interfere with the regulatory roles of NRBP1 and NRBP2 in L1 mobility. Shown is one representative picture of the colony assay to examine L1 mobility. (F) NRB and dimerization region of NRBP2 are essential for its inhibitory role on L1. Shown is one representative picture of the colony assay depicted in . (G) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is one representative picture of the colony assay depicted in . (H) Both C- and N-terminal halves of NRBP2 interact with ORF1 in HEK293T cells. (I) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is one representative picture of the colony assay depicted in .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Western Blot, Expressing, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Schematic diagrams of NRBP2 and the NRBP2 mutants used in this study. NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (B) The NRB and dimerization region of NRBP2 are required for NRBP2 to inhibit L1 retrotransposition. Quantification of L1 activity is shown in the top panel. N = 5 biological replicates. One representative Western blot showing expression of the respective NRBP2 mutant variants is shown in the bottom panel. (C) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is quantification of L1 activity upon expression of the respective NRBP2 variants. N = 3 biological replicates. (D) Schematic diagram of NRBP1 and the NRBP1 halves used in this study. LCR: low complexity region; NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (E) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is quantification of L1 activity upon expression of the respective NRBP1 variants. For NRBP1, N = 5 biological replicates. For NRBP1-N and NRBP1-C, N = 3 biological replicates. (F) The C-terminal half of NRBP2 promotes enrichment of NRBP1 to the peripheral region of HeLa cells. White arrows point to NRBP1 without NRBP2 transfection. Yellow arrows indicate cells with enrichment of NRBP1 to the peripheral region upon NRBP2 overexpression. Scale bar 10 µm. (G) The C-terminal half of NRBP1 promotes enrichment of full-length NRBP1 to the peripheral region of HeLa cells. Scale bar 10 µm. (H) Overexpressing NRBP2 and C-terminal half of NRBP1 or NRBP2 reduces endogenous NRBP1 protein level in HeLa cells. Shown is one representative Western blot to detect the respective NRBP1 or NRBP2 variants and endogenous NRBP1. N = 3 biological replicates. See also , and .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, Mutagenesis, Transfection, Over Expression

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Proteasome inhibitor MG132 abolishes capability of NRBP2 and the C-terminal halves of NRBP1 and NRBP2 to reduce protein level of NRBP1. Shown is quantification of relative protein levels of NRBP1 in HeLa cells. GAPDH was used for normalization. N = 3 biological replicates. (B) Proteasome inhibitor MG132 prevents enrichment of NRBP1 to the peripheral region of HeLa cells upon overexpressing the C-terminal half of NRBP1 or NRBP2. Shown are representative confocal images of immunofluorescence stained cells. Scale bar 10 µm. (C) NRB motif in NRBP2 is essential to reduce protein level of NRBP1 in HeLa cells. The upper panel shows the quantification of the relative NRBP1 protein level normalized by GAPDH. Bottom panel is one representative Western blot used for quantification. N = 3 biological replicates. (D) NRB motifs in C-terminal half of NRBP1 are essential to inhibit L1 and to reduce the protein level of NRBP1. Quantification of L1 activity in HeLa cells is shown in the top panel. N = 3 biological replicates. One representative Western blot showing expression of the respective proteins is shown in the bottom panel. (E) The C-terminal half of NRBP1 interacts with the full-length NRBP1 in an NRB-dependent manner. (F) The C-terminal half of NRBP2 interacts with the full-length NRBP1 in an NRB-dependent manner. (G) Lack of NRB and dimerization region does not prevent interaction between NRBP2 and NRBP1. (H) Both the N- and C-terminal halves of NRBP2 interact with NRBP1. For E-H , HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Flag-NRBP1. The co-precipitated proteins were detected by using HA or Myc antibody. See also and S7.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay, Expressing, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 reduce protein level of full-length NRBP1 in HeLa cells and this could be blocked with MG132 treatment. Shown is one representative Western blot result used for the quantification in the main . (B) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 promote NRBP1 decay independently of ELOB and ELOC. In line with prior findings, reducing either ELOB or ELOC resulted in a decrease in the protein levels of the other. , Shown is one representative Western blot result used for the quantification in (C) . (C) Quantification of three independent Western blot experiments from (B) . (D) The NRB motifs in the C-terminal half of NRBP1 are essential to inhibit L1. Shown is one representative picture of the colony assay depicted in the main .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Western Blot, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A and B) Pie charts illustrating the numbers of genes displaying significantly altered expression levels (fold change ≥ 1.5 and FDR < 0.05) following the knockdown of either NRBP2 (A) or NRBP1 (B) in HeLa cells. (C) Venn Diagram showing overlapping genes upregulated by NRBP2 knockdown in HeLa cells and genes activated upon L1 overexpression in RPE cells. p < 2.081e-25. The p-value was calculated using http://nemates.org/MA/progs/overlap_stats.html . to determine the statistical significance of the overlap between the two groups of genes. (D) GO term analysis for the overlapping genes in (C) with the online software DAVID. Pathways exhibiting an enrichment with -log 10 p-value >2 are presented. (E) qRT-PCR to confirm activation of selected innate immunity genes upon NRBP2 knockdown in HeLa cells. N = 3 biological replicates. (F) mRNA levels of NRBP1 and NRBP2 in samples obtained from both healthy individuals and RA patients. Data is taken from Autoimmune Diseases Explorer ( https://adex.genyo.es ). See also Table S2.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Expressing, Knockdown, Over Expression, Software, Quantitative RT-PCR, Activation Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Maximum likelihood phylogeny of the IQ-TREE analysis comprising 2,065 quality-filtered NRBP proteins belonging to 663 species and including 417 amino acid positions. Orthologues of human NRBP2 are recovered as a monophyletic group with perfect bootstrap support (blue). All NRBP2 belong to the Euteleostomi. Its sister clade contains all orthologues of human NRBP1 derived from vertebrate taxa (orange) and has perfect bootstrap support as well. The NRBP sequence of the sponge Amphimedon queenslandica was chosen as outgroup taxon. Branch lengths are amino acid substitutions per site. (B) The distance from leaf to root was determined in (A) for all NRBP proteins. The NRBP2 clade accumulated considerably more amino acid substitutions than the NRBP1 clade and the average invertebrate NRBP protein. A consequence of this is that all invertebrate NRBP proteins are more similar to human NRBP1 than they are to human NRBP2. (C) Scatter plot of BlastP bit scores resulting from 462 comparisons of invertebrate NRBP either to human NRBP1 or to human NRBP2. For all value pairs, the bit score for the NRBP1 comparison is higher than that of the respective comparison to NRBP2. (D) Plot and paired t-test for the values described in (C) results in a p-value of 1.5 × 10 -202 . This indicates that human NRBP1 is more similar to invertebrate NRBP than human NRBP2 is. See also .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Derivative Assay, Sequencing, Comparison

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) We mapped 2,065 homologs of human NRBP1/2 to a phylogenetic tree of eukaryotes. Clades drawn with red lines indicate the presence of NRBP , whereas clades drawn with black lines indicate the absence of NRBP . NRBP is present in the early metazoan branches Porifera and Placozoa, but it is absent from all non-metazoan eukaryotes. The figure is a modified image from . The original image is licensed under the Creative Commons Attribution 2.5 Generic license ( https://creativecommons.org/licenses/by/2.5/deed.en ). (B) We mapped 669 candidate orthologues of human NRBP2 to a phylogenetic tree of vertebrates. Clades drawn with red lines indicate the presence of NRBP2 , whereas clades drawn with black lines indicate the absence of NRBP2 . All NRBP2 map either to the Sarcopterygii or to the Actinopterygii, indicating an evolutionary origin of NRBP2 either in the Eutelestomi, or earlier in vertebrate evolution. All presented clades (black and red lines) have candidate orthologues of NRBP1 . Remarkably, clades with NRBP2 have a ‘Type II’ L1 ORF1, which is characterised by ‘transposase 22’, whereas the clades lacking NRBP2 also lack the transposase 22. For details, please refer to the discussion section. The image is modified from https://en.wikipedia.org/wiki/Actinopterygii .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Modification

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) The NRBP1/2 antibody used for MS interactome analysis recognizes both NRBP1 and NRBP2. (B) Both NRBP1 and NRBP2 are associated with multiple known L1 interactors or regulators. HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Myc-Flag-NRBP1 or Myc-Flag-NRBP2, co-precipitated endogenous proteins were detected by using respective antibodies. Myc-Flag-NRBP1 and Myc-Flag-NRBP2 were detected with Myc antibody.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Scatter plots showing proteins significantly enriched (fold change ≥ 5, p -value < 0.05) in NRBP1/2 IP vs. IgG in MCF-7 cells. NRBP1 and NRBP2 are shown in red. Previously known NRBP1 interactors are labelled in blue. ORF1 is marked in orange. (B) Endogenous ORF1 is co-immunoprecipitated with NRBP1 and/or NRBP2. An antibody recognizing both NRBP1 and NRBP2 was used to pull down endogenous NRBP1 and NRBP2 in MCF-7 cells. Co-precipitated ORF1 was detected by using ORF1 antibody. (C) Endogenous ORF1 is co-immunoprecipitated with both NRBP1 and NRBP2. MCF-7 cells were transfected with either Myc-Flag-tagged NRBP1 or Myc-Flag-tagged NRBP2. The cell lysates were incubated with Flag antibody and the immunoprecipitated proteins were detected by Western blot with the indicated antibodies. (D) NRBP1 and NRBP2 are co-immunoprecipitated with ORF1. Flag antibody was used to pull down ORF1-Flag, and Myc antibody was used to detect co-immunoprecipitated Myc-NRBP1 and Myc-NRBP2 in HeLa cells. (E-L) NRBP1 activates and NRBP2 inhibits L1 retrotransposition. One representative L1 retrotransposition activity is shown in (E) , (G) , (I) and (K) . Quantitation of L1 retrotransposition activity is shown in the top panels of (F) , (H) , (J) and (L) . Western blots indicating successful overexpression or knockdown are shown in the bottom panels of (F) , (H) , (J) and (L) . N = 4 biological replicates for (E) and (F) . N = 3 biological replicates for (G) and (H) . N = 7 biological replicates for (I) and (J) . N = 3 biological replicates for (K) and (L) . See also and Table S1.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Immunoprecipitation, Transfection, Incubation, Western Blot, Activity Assay, Quantitation Assay, Over Expression, Knockdown

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) MCF-7 cells show higher ORF1 protein level than HeLa cells. (B) Western blot assesses efficiency of NRBP1 knockdown and ORF1 IP for the experiments in . (C) Induction of ORF1-Flag enriched foci by NRBP2 overexpression is independent of G3BP1. HA-NRBP2, ORF1-Flag and G3BP1 were stained by HA, Flag and G3BP1 antibodies, respectively. Scale bar 10 µm. (D) Inhibition of L1 retrotransposition by NRBP2 is independent of G3BP1. Quantification of the colony assay is shown in the top panel and one representative Western blot result is shown in the bottom panel. N = 4. (E) One representative picture of the colony assay depicted in (D) . (F) Western blot assesses efficiency of HA-NRBP2 transfection and ORF1 IP for the experiments in .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Western Blot, Knockdown, Over Expression, Staining, Inhibition, Colony Assay, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown results in an enrichment of ORF1 in cytoplasmic foci in MCF-7 cells. The yellow arrows point to the ORF1 foci. Shown are immunofluorescence staining of endogenous ORF1 and G3BP1 proteins with their respective antibodies. Scale bar 10 µm. (B) NRBP1 knockdown impairs colocalization of ORF1 and L1 mRNA. Shown are immunofluorescence staining of endogenous ORF1 protein with ORF1 antibody (green) and L1 mRNAs detected via smFISH with L1 probes (violet). Nucleus was stained with Hoechst 33342 (blue). The arrows in the top panel point to co-localized ORF1 and L1 mRNA, the arrows in the bottom panel point to the ORF1-enriched puncta without an enrichment of L1 mRNA. Scale bar 10 µm. (C) ORF1 RIP-qPCR to quantitate ORF1-associated L1 mRNA. Three primer pairs targeting to L1 5′ UTR, ORF1 and ORF2 were used for qPCR. N = 3 biological replicates. (D) HA-NRBP2 overexpression results in an enrichment of ORF1-Flag in cytoplasmic puncta in HeLa cells. The yellow arrows point to colocalized ORF1 and G3BP1 foci. Scale bar 10 µm. (E) NRBP2 overexpression prevents the interaction between ORF1 protein and L1 mRNA. RIP-qPCR was carried out to quantitate Flag-tagged ORF1 co-immunoprecipitated L1 mRNA levels with indicated primer pairs. N = 3 biological replicates. See also .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Knockdown, Immunofluorescence, Staining, Over Expression, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Overexpressing NRBP1 or NRBP2 does not interfere with binding of their ortholog counterpart to ORF1. Co-IP was performed in HeLa cells by using Flag antibody. (B) NRBP1 knockdown abolishes inhibitory effect of NRBP2 on L1 retrotransposition. Shown is a representative picture of the colony assay depicted in . (C) NRBP2 knockdown does not significantly increase mRNA level of NRBP1. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates. (D) NRBP1 knockdown increases protein level of NRBP2. Shown is quantification of four replicates in . (E) NRBP1 knockdown increases mRNA level of NRBP2. Shown is quantification of relative mRNA levels of NRBP1 and NRBP2 normalized by GAPDH in HeLa cells. N = 3 biological replicates.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) NRBP1 knockdown abolishes the inhibitory effect of NRBP2 on L1 in HeLa cells. Shown is quantitation of L1 retrotransposition activity. N = 4 biological replicates. (B) Overexpression of NRBP2-Myc reduces endogenous NRBP1 protein level. Shown is one representative Western blot using the samples from (A) to detect endogenous NRBP1 and transfected NRBP2-Myc. (C and D) NRBP1 and NRBP2 negatively affect the protein levels of each other in HeLa cells. Endogenous NRBP1 and NRBP2 were detected using NRBP1/2 antibody. Shown is one representative Western blot result (C) . Quantification of the protein levels was shown in (D) . N = 4 biological replicates. (E) Overexpressing NRBP2-Myc results in an enrichment of HA-NRBP1 to the peripheral region of HeLa cells. Yellow arrows point to cells co-expressing HA-NRBP1 and NRBP2-Myc. White arrow indicates a cell only expressing NRBP1. HA-NRBP1 and NRBP2-Myc were detected using HA and Myc antibodies. Scale bar 10 µm. See also .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Knockdown, Quantitation Assay, Activity Assay, Over Expression, Western Blot, Transfection, Expressing

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Diagram illustrations of domains / motifs in human NRBP1 and NRBP2 proteins. (B) NRBP1 structure prediction by AlphaFold (AF-Q9UHY1-F1) reveals a potential unstructured N-terminal LCR. (C) A simple schematic diagram of NRBP1, NRBP2 and their mutants for the LCR-swapping experiment. (D) A representative Western blot to show expression of NRBP1, NRBP2 and their mutants in (C) and (E) . (E) LCR-swapping does not interfere with the regulatory roles of NRBP1 and NRBP2 in L1 mobility. Shown is one representative picture of the colony assay to examine L1 mobility. (F) NRB and dimerization region of NRBP2 are essential for its inhibitory role on L1. Shown is one representative picture of the colony assay depicted in . (G) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is one representative picture of the colony assay depicted in . (H) Both C- and N-terminal halves of NRBP2 interact with ORF1 in HEK293T cells. (I) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is one representative picture of the colony assay depicted in .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Western Blot, Expressing, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Schematic diagrams of NRBP2 and the NRBP2 mutants used in this study. NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (B) The NRB and dimerization region of NRBP2 are required for NRBP2 to inhibit L1 retrotransposition. Quantification of L1 activity is shown in the top panel. N = 5 biological replicates. One representative Western blot showing expression of the respective NRBP2 mutant variants is shown in the bottom panel. (C) The C-terminal half of NRBP2 is necessary and sufficient to inhibit L1. Shown is quantification of L1 activity upon expression of the respective NRBP2 variants. N = 3 biological replicates. (D) Schematic diagram of NRBP1 and the NRBP1 halves used in this study. LCR: low complexity region; NES: nuclear export signal; NLS: nuclear localization signal; BC box: Elongin BC-binding motif; NRB: nuclear receptor-binding motif. (E) The C-terminal half of NRBP1 functions oppositely to the full-length NRBP1 and inhibits L1. Shown is quantification of L1 activity upon expression of the respective NRBP1 variants. For NRBP1, N = 5 biological replicates. For NRBP1-N and NRBP1-C, N = 3 biological replicates. (F) The C-terminal half of NRBP2 promotes enrichment of NRBP1 to the peripheral region of HeLa cells. White arrows point to NRBP1 without NRBP2 transfection. Yellow arrows indicate cells with enrichment of NRBP1 to the peripheral region upon NRBP2 overexpression. Scale bar 10 µm. (G) The C-terminal half of NRBP1 promotes enrichment of full-length NRBP1 to the peripheral region of HeLa cells. Scale bar 10 µm. (H) Overexpressing NRBP2 and C-terminal half of NRBP1 or NRBP2 reduces endogenous NRBP1 protein level in HeLa cells. Shown is one representative Western blot to detect the respective NRBP1 or NRBP2 variants and endogenous NRBP1. N = 3 biological replicates. See also , and .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, Mutagenesis, Transfection, Over Expression

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Proteasome inhibitor MG132 abolishes capability of NRBP2 and the C-terminal halves of NRBP1 and NRBP2 to reduce protein level of NRBP1. Shown is quantification of relative protein levels of NRBP1 in HeLa cells. GAPDH was used for normalization. N = 3 biological replicates. (B) Proteasome inhibitor MG132 prevents enrichment of NRBP1 to the peripheral region of HeLa cells upon overexpressing the C-terminal half of NRBP1 or NRBP2. Shown are representative confocal images of immunofluorescence stained cells. Scale bar 10 µm. (C) NRB motif in NRBP2 is essential to reduce protein level of NRBP1 in HeLa cells. The upper panel shows the quantification of the relative NRBP1 protein level normalized by GAPDH. Bottom panel is one representative Western blot used for quantification. N = 3 biological replicates. (D) NRB motifs in C-terminal half of NRBP1 are essential to inhibit L1 and to reduce the protein level of NRBP1. Quantification of L1 activity in HeLa cells is shown in the top panel. N = 3 biological replicates. One representative Western blot showing expression of the respective proteins is shown in the bottom panel. (E) The C-terminal half of NRBP1 interacts with the full-length NRBP1 in an NRB-dependent manner. (F) The C-terminal half of NRBP2 interacts with the full-length NRBP1 in an NRB-dependent manner. (G) Lack of NRB and dimerization region does not prevent interaction between NRBP2 and NRBP1. (H) Both the N- and C-terminal halves of NRBP2 interact with NRBP1. For E-H , HEK293T cells were transfected with the indicated plasmids. Flag antibody was used to pull down Flag-NRBP1. The co-precipitated proteins were detected by using HA or Myc antibody. See also and S7.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay, Expressing, Transfection

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 reduce protein level of full-length NRBP1 in HeLa cells and this could be blocked with MG132 treatment. Shown is one representative Western blot result used for the quantification in the main . (B) Both the full-length NRBP2 and the C-terminal half of NRBP1 or NRBP2 promote NRBP1 decay independently of ELOB and ELOC. In line with prior findings, reducing either ELOB or ELOC resulted in a decrease in the protein levels of the other. , Shown is one representative Western blot result used for the quantification in (C) . (C) Quantification of three independent Western blot experiments from (B) . (D) The NRB motifs in the C-terminal half of NRBP1 are essential to inhibit L1. Shown is one representative picture of the colony assay depicted in the main .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Western Blot, Colony Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A and B) Pie charts illustrating the numbers of genes displaying significantly altered expression levels (fold change ≥ 1.5 and FDR < 0.05) following the knockdown of either NRBP2 (A) or NRBP1 (B) in HeLa cells. (C) Venn Diagram showing overlapping genes upregulated by NRBP2 knockdown in HeLa cells and genes activated upon L1 overexpression in RPE cells. p < 2.081e-25. The p-value was calculated using http://nemates.org/MA/progs/overlap_stats.html . to determine the statistical significance of the overlap between the two groups of genes. (D) GO term analysis for the overlapping genes in (C) with the online software DAVID. Pathways exhibiting an enrichment with -log 10 p-value >2 are presented. (E) qRT-PCR to confirm activation of selected innate immunity genes upon NRBP2 knockdown in HeLa cells. N = 3 biological replicates. (F) mRNA levels of NRBP1 and NRBP2 in samples obtained from both healthy individuals and RA patients. Data is taken from Autoimmune Diseases Explorer ( https://adex.genyo.es ). See also Table S2.

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Expressing, Knockdown, Over Expression, Software, Quantitative RT-PCR, Activation Assay

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) Maximum likelihood phylogeny of the IQ-TREE analysis comprising 2,065 quality-filtered NRBP proteins belonging to 663 species and including 417 amino acid positions. Orthologues of human NRBP2 are recovered as a monophyletic group with perfect bootstrap support (blue). All NRBP2 belong to the Euteleostomi. Its sister clade contains all orthologues of human NRBP1 derived from vertebrate taxa (orange) and has perfect bootstrap support as well. The NRBP sequence of the sponge Amphimedon queenslandica was chosen as outgroup taxon. Branch lengths are amino acid substitutions per site. (B) The distance from leaf to root was determined in (A) for all NRBP proteins. The NRBP2 clade accumulated considerably more amino acid substitutions than the NRBP1 clade and the average invertebrate NRBP protein. A consequence of this is that all invertebrate NRBP proteins are more similar to human NRBP1 than they are to human NRBP2. (C) Scatter plot of BlastP bit scores resulting from 462 comparisons of invertebrate NRBP either to human NRBP1 or to human NRBP2. For all value pairs, the bit score for the NRBP1 comparison is higher than that of the respective comparison to NRBP2. (D) Plot and paired t-test for the values described in (C) results in a p-value of 1.5 × 10 -202 . This indicates that human NRBP1 is more similar to invertebrate NRBP than human NRBP2 is. See also .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Derivative Assay, Sequencing, Comparison

Journal: bioRxiv

Article Title: Targeting the paralog protein for degradation accounts for the opposite roles of NRBP1 and NRBP2 in regulating LINE1 retrotransposition

doi: 10.1101/2024.06.14.598964

Figure Lengend Snippet: (A) We mapped 2,065 homologs of human NRBP1/2 to a phylogenetic tree of eukaryotes. Clades drawn with red lines indicate the presence of NRBP , whereas clades drawn with black lines indicate the absence of NRBP . NRBP is present in the early metazoan branches Porifera and Placozoa, but it is absent from all non-metazoan eukaryotes. The figure is a modified image from . The original image is licensed under the Creative Commons Attribution 2.5 Generic license ( https://creativecommons.org/licenses/by/2.5/deed.en ). (B) We mapped 669 candidate orthologues of human NRBP2 to a phylogenetic tree of vertebrates. Clades drawn with red lines indicate the presence of NRBP2 , whereas clades drawn with black lines indicate the absence of NRBP2 . All NRBP2 map either to the Sarcopterygii or to the Actinopterygii, indicating an evolutionary origin of NRBP2 either in the Eutelestomi, or earlier in vertebrate evolution. All presented clades (black and red lines) have candidate orthologues of NRBP1 . Remarkably, clades with NRBP2 have a ‘Type II’ L1 ORF1, which is characterised by ‘transposase 22’, whereas the clades lacking NRBP2 also lack the transposase 22. For details, please refer to the discussion section. The image is modified from https://en.wikipedia.org/wiki/Actinopterygii .

Article Snippet: Myc-DDK-NRBP1 (pBY4284) and Myc-DDK-NRBP2 (pBY4285) were generated by inserting NRBP1 and NRBP2 PCR products between the AscI / NotI sites of the pCMV6-AN-Myc-DDK vector (Origene).

Techniques: Modification

Journal: Particle and Fibre Toxicology

Article Title: Mechanisms of nanosized titanium dioxide-induced testicular oxidative stress and apoptosis in male mice

doi: 10.1186/s12989-014-0047-3

Figure Lengend Snippet: Real time PCR primer pairs

Article Snippet: The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of SOD, CAT, GPx, GST, GR, Cyp1b1, Car3, caspase-3, Bcl-2, Nrbp2, Acaa2, Axud1, and cytochrome c were calculated from a standard curve for each sample.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Particle and Fibre Toxicology

Article Title: Mechanisms of nanosized titanium dioxide-induced testicular oxidative stress and apoptosis in male mice

doi: 10.1186/s12989-014-0047-3

Figure Lengend Snippet: Real time PCR primer pairs

Article Snippet: To determine protein levels of SOD, CAT, GPx, GST, GR, Cyp1b1, Car3, caspase-3, Bcl-2, Nrbp2, Acaa2, Axud1, and cytochrome c in the testes, total protein from the frozen testicular tissues ( N = 5 in each group) from experimental and control mice was extracted using Cell Lysis Kits (GENMED SCIENTIFICS INC.USA) and quantified using BCA protein assay kits (GENMED SCIENTIFICS INC.USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: bioRxiv

Article Title: Pseudokinase HPO-11 inhibits nonsense-mediated decay to ensure genome stability in C. elegans

doi: 10.1101/2022.09.04.506508

Figure Lengend Snippet: FLAG-HPO-11 interactome a. The difference of log 2 -transformed mean LFQ intensities of FLAG-HPO-11 IP and control IP was plotted against the negative log 10 p-value of a one-sided two-sample t-test (n=3). Proteins with a p-value below 0.05 and a student’s t-test difference of at least 2 were considered as significantly enriched candidates (light grey). The bait protein HPO-11 is shown in green. Homologs of known NRBP1 interactors (ELB-1, ELC-1 and Y48C3A.12) are depicted in dark grey, SMG-2 in orange. b. Gene ontology term enrichment analysis for the interactors of HPO-11. Selected enriched GO terms are shown with significance.

Article Snippet: Anti-Flag (F3165, Sigma-Aldrich), anti-V5-Tag antibody (ab27671, Abcam), anti-GFP antibody (ab290, Abcam), Anti-DNA-RNA hybrid antibody S9.6 (MABE1095, Sigma-Aldrich), anti-NRBP1/2 antibody (21549-1-AP, Proteintech Europe), Phospho-Histone γH2AX antibody (Ser139) (9718, Cell Signaling Technology) and GAPDH (ab8245, Abcam).

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: Pseudokinase HPO-11 inhibits nonsense-mediated decay to ensure genome stability in C. elegans

doi: 10.1101/2022.09.04.506508

Figure Lengend Snippet: h p o -11 and its human homologs NRBP1 and NRBP2 share conserved functions a. NRBP1/2 interact with UPF1 in MCF-7 cells. Shown is one representative result of three biological replicates. b. siRNA knock-down of either NRBP1 or NRBP2 caused DNA damage response. Shown is a western blot of γ-H2AX in HEK293T cell, N = 3 biological replicates.

Article Snippet: Anti-Flag (F3165, Sigma-Aldrich), anti-V5-Tag antibody (ab27671, Abcam), anti-GFP antibody (ab290, Abcam), Anti-DNA-RNA hybrid antibody S9.6 (MABE1095, Sigma-Aldrich), anti-NRBP1/2 antibody (21549-1-AP, Proteintech Europe), Phospho-Histone γH2AX antibody (Ser139) (9718, Cell Signaling Technology) and GAPDH (ab8245, Abcam).

Techniques: Western Blot

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: Primers for RT-qPCR

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques:

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: Poor expression of NRBP2 in TC cells indicates unfavorable prognosis in patients. (a), differentially expressed genes between TC and normal samples in the GSE165724 dataset; (b), NRBP2 expression predicted in TCGA-THCA; (c), IHC data of NRBP2 predicted via the HPA system; (d), mRNA expression of NRBP2 in tissues from 71 TC patients examined by RT-qPCR; (e), representative IHC images of NRBP2 and staining intensity in tumor tissues (PTC) and the adjacent normal from patients; (f)-(g), mRNA (f) and protein (g) levels of NRBP2 in Nthy-ori 3–1 cells and TC cell lines (BCPAP, TPC-1, CAL62 and SW579) examined by RT-qPCR and western blot analysis. Three repetitions were performed. ** p < 0.01.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Expressing, Quantitative RT-PCR, Staining, Western Blot

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: Overexpression of NRBP2 reduces activity of TC cells in vitro . A-B, mRNA (a) and protein (b) levels of NRBP2 in TPC-1 and CAL62 cells after oe-NRBP2 transfection examined by RT-qPCR and western blot assays; (c), DNA replication ability of TPC-1 and CAL62 cells examined by the EdU labeling assay; (d), colony formation ability of TPC-1 and CAL62 cells determined by the colony formation assay; (e), apoptosis rate in TPC-1 and CAL62 cells determined by flow cytometry. Three repetitions were performed. ** p < 0.01.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Over Expression, Activity Assay, In Vitro, Transfection, Quantitative RT-PCR, Western Blot, Labeling, Colony Assay, Flow Cytometry

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: NRBP2 reduces expression of TME markers and angiogenesis in TC cells. (a), IL-6 and VEGFA concentrations in TPC-1 and CAL62 cell secretions examined using ELISA kits; (b), M1/M2 polarization of macrophages cells co-cultured with TPC-1 and CAL62 cells examined by flow cytometry; (c), expression of the M2-polarized macrophage marker Arg1 in the macrophages determined by immunofluorescence staining; (d), angiogenesis ability of HUVECs co-cultured with TPC-1 and CAL62 cells analyzed by tube formation assay. Three repetitions were performed. ** p < 0.01 vs. oe-NC.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Marker, Immunofluorescence, Staining, Tube Formation Assay

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: NRBP2 reduces TC tumorigenesis and M2 macrophage infiltration in vivo . (a), growth rate of xenograft tumors formed by TPC-1 and CAL62 cells in nude mice; (b) tumor weight on day 36; (c)-(e), expression of KI67, Arg1 and CD31 in xenograft tumor tissues examined by IHC. In each group, n = 6. ** p < 0.01 vs. oe-NC.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: In Vivo, Expressing

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: NRBP2 is negatively regulated by GATA1 . (a), candidate transcription factors that might bind to the NRBP2 promoter predicted in JASPAR; the numbers in the square frame indicates the binding sites; the gene names are presented on the frames and their corresponding putative binding sequences with NRBP2 promoter are provided below the frame; (b), binding relationship between EGR1, DMRTC2, GATA6, GATA1, FOXC2 and CEBPB with the promoter of NRBP2 validated through ChIP-qPCR assays; (c), an inverse correlation between GATA1 and NRBP2 expression in TCGA-THCA; (d), GATA1 expression in the tumor and the adjacent tissues from 71 TC patients detected by RT-qPCR; (e), an inverse correlation between GATA1 and NRBP2 expression in the collected tumor tissues by Spearman’s correlation analysis; F-G, mRNA (f) and protein (g) levels of NRBP2 in Nthy-ori 3–1 cells and TC cell lines (BCPAP, TPC-1, CAL62 and SW579) detected by RT-qPCR and western blot assays; (h), pGL3 vector containing the NRBP2 promoter sequence constructed for luciferase assay; I, luciferase activity of the luciferase vector in 293 T cells; J-K, mRNA (j) and protein (k) levels of NRBP2 in TPC-1 and CAL62 cells after oe-GATA1 transfection detected by RT-qPCR; (l), GATA1 protein level in WT or GATA1-deleted cells examined by western blot analysis; (m), GATA1 protein level in cells after further oe-GATA1 transfection examined by western blot analysis; N, NRBP2 protein level in cells examined by western blot analysis. Three repetitions were performed. ** p < 0.01.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Sequencing, Construct, Luciferase, Activity Assay, Transfection

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: GATA1 recruits HDAC2 to suppress NRBP2 expression. (a), significant H3K9ac in the NRBP2 promoter predicted in the UCSC browser; (b), H3K9ac level in three pairs of TC tumor tissues and the adjacent tissues examined by the ChIP-qPCR assay; (c)-(d), mRNA (c) and protein (d) levels of NRBP2 in TPC-1 and CAL62 cells after Tacedinaline treatment detected by RT-qPCR and western blot assay; (e), HDACs that can bind to GATA1 in TPC-1 cells validated by the Co-IP assays; input refers to the positive control that confirm the corresponding proteins are expressed in cells; IgG represents the negative control; (f), luciferase activity of the luciferase vector in 293 T cells. Three repetitions were performed; G-H, mRNA (g) and protein (h) expression of NRBP2 in cells after CAY10683 treatment examined by RT-qPCR and western blot analysis; (i), mRNA expression of GATA1, HDAC2 , and NRBP2 in cells after sh-GATA1 transfection determined by RT-qPCR. ** p < 0.01.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Positive Control, Negative Control, Luciferase, Activity Assay, Plasmid Preparation, Transfection

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: GATA1 or HDAC2 overexpression counteracts the inhibitory effect of oe-NRBP2 on TC cells. A-B, protein level of NRBP2 in TPC-1 (a) and CAL62 (b) cells after GATA1 or HDAC2 overexpression, respectively, examined by western blot assays; (c), proliferation activity of TPC-1 and CAL62 cells examined by the EdU labeling assay; (d), apoptosis rate in TPC-1 and CAL62 cells determined by flow cytometry; (e), IL-6 and VEGFA concentrations in TPC-1 and CAL62 cell secretions examined using ELISA kits; (f)-(g), polarization of M2 macrophages cells co-cultured with TPC-1 and CAL62 cells determined by flow cytometry and immunofluorescence staining; (h), angiogenesis ability of HUVECs co-cultured with TPC-1 and CAL62 cells determined by tube formation assay. Three repetitions were performed. ** p < 0.01.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Over Expression, Western Blot, Activity Assay, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunofluorescence, Staining, Tube Formation Assay

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: Graphical abstract. In TC, GATA1 recruits HDAC2 to the promoter region of NRBP2 to induce NRBP2 transcriptional suppression by deacetylation, which leads to increased M2 polarization of macrophages as well as increased angiogenesis, proliferation, and tumor development.

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques:

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: Correlation between NRBP2 expression and the clinical characteristics of patients

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Expressing

Journal: Bioengineered

Article Title: GATA binding protein 1 recruits histone deacetylase 2 to the promoter region of nuclear receptor binding protein 2 to affect the tumor microenvironment and malignancy of thyroid carcinoma

doi: 10.1080/21655979.2022.2068921

Figure Lengend Snippet: Clinical characteristics of included patients with TC

Article Snippet: The promoter sequence of NRBP2 was obtained from UCSC and sub-cloned to the pGL3 luciferase vector (E1751, Promega, Fitchburg, WI, USA).

Techniques: Expressing