Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 translocates to depolarized mitochondria in a Parkin-dependent manner. ( A ) HeLa cells transfected with expression constructs for FLAG-UBXD1, mitoYFP, and Parkin were treated with 10, 15, 25, 40, or 50 µM CCCP for 6 hours or left untreated as control. Cells were fixed, stained using mouse anti-FLAG antibodies to detect UBXD1 and analyzed by confocal microscopy. ( B ) HeLa cells transfected with expression constructs for FLAG-UBXD1, mitoYFP, and Parkin or vector control were treated with 50 µM CCCP for six hours of left untreated as controls. Cells were analyzed as above.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Construct, Staining, Confocal Microscopy, Plasmid Preparation

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: Mitochondrial translocation of UBXD1 in HEK293 cells and during Parkin-independent mitophagy. ( A ) HEK293 cells transfected with expression constructs for FLAG-UBXD1 and mitoYFP were treated for 6 hours with 50 µM CCCP or left untreated as controls. After fixation and anti-FLAG staining, cells were analyzed by confocal microscopy. Fluorescence intensities of FLAG-UBXD1 (red) and mitoYFP (green) along the white line are plotted. Shown are representative images of three independent experiments. ( B ) HeLa cells transfected with expression constructs for FLAG-UBXD1 and mitoYFP were treated for 24 hours with 1 mM DFP or left untreated as control. Cells were fixed, stained using mouse anti-FLAG antibodies to detect UBXD1 and analyzed by confocal microscopy. ( C ) HeLa cells transfected with expression constructs for GFP-LC3 and mitoDsRed were treated for 24 hours with 1 mM DFP, treated for 6 hours with 100 nM bafilomycin or left untreated, fixed and analyzed by confocal microscopy.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: VIM and PUB, but not the UBX domain of UBXD1 are dispensable for mitochondrial translocation during mitophagy. ( A ) HeLa cells transfected with mitoYFP-T2A-Parkin-myc3 and FLAG-UBXD1, FLAG-UBXD1ΔPUB, FLAG-UBXD1ΔVIM, FLAG-UBXD1ΔUBX, or FLAG-UBXonly expression constructs were treated with CCCP for 6 hours or left untreated as control, stained using mouse anti-FLAG antibodies and analyzed by confocal microscopy. ( B ) The ratio of mitochondrial to total FLAG-UBXD1 or various FLAG-UBXD1 variants as measure for mitochondrial translocation was quantified by image analysis of confocal pictures obtain from cells treated as in A. Shown are box plots of three independent experiments with at least 15 cells per experiment and condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. *** denotes p-values < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Construct, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 recruits p97 to mitochondria under mitophagic conditions. ( A ) HeLa cells transfected with expression plasmids for FLAG-UBXD1 and mitoYFP-T2A-Parkin-myc3 were treated with CCCP for 6 hours or left untreated as control. Cells were fixed, stained using rabbit anti-FLAG and mouse anti-p97 antibodies and analyzed by confocal microscopy. ( B ) HeLa cells transfected with expression plasmids for FLAG-UBXD1 or vector control and mitoYFP-T2A-Parkin-myc3 were treated as in A. To quantify p97 redistribution to mitochondria, the ratio of mitochondrial p97 to total p97 was determined by image analysis of confocal images. Shown are box plots of three independent experiments with at least 15 cells per experiment and condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. ***Denotes p-values < 0.001.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Plasmid Preparation

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: The UBX domain of UBXD1 is essential for mitochondrial translocation of p97. HeLa cells transfected with expression plasmids for FLAG-UBXD1 or variants of FLAG-UBXD1 and mitoYFP-T2A-Parkin-myc3 were treated with CCCP for 6 hours or left untreated as control. Fixed cells were stained using rabbit anti-FLAG and mouse anti-p97 antibodies and analyzed by confocal microscopy. The box plots represent three independent experiments with at least 15 cells/experiment/condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. *denotes p-values < 0.05, ***p-values < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: The VIM domain of UBXD1 is not involved in mitochondrial translocation and p97 recruitment. HeLa cells transfected with expression plasmid for FLAG-VIMonly and mitoYFP-T2A-Parkin-myc3 were treated with CCCP for 6 hours or left untreated as control. Fixed cells were stained using rabbit anti-FLAG and mouse anti-p97 antibodies and analyzed by confocal microscopy. The box plots represent three independent experiments with at least 15 cells/experiment/condition. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Translocation Assay, Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: HeLa cells transfected with expression plasmids for mitochondria-targeted dsRED (mitodsRED) and YFP-UBXD1 or YFP fused to the mitochondrial membrane targeting signal ActA were fixed, stained using mouse anti-p97 antibodies, and analyzed by confocal microscopy. Shown are representative images out of three independent experiments. The box plots represent three independent experiments with at least 15 cells/experiment/condition. Statistical significance was assessed by by Student’s t-test. ***denotes p < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 promotes mitophagy. ( A ) HeLa cells transfected with expression plasmids for FLAG-UBXD1 or vector control, mcherry-Parkin and GFP-LC3 were fixed, stained using mouse anti-FLAG antibodies, and analyzed by confocal microscopy. ( B ) HeLa cells transfected with expression plasmids for UBXD1 or vector control and mKeima-T2A-Parkin-myc3 were treated for 12 hours with CCCP or left untreated and analyzed by flow cytometry. Shown are representative density plots (left panels). The box plot represents 6 independent experiments with in total 11 technical replicates. Statistical significance was assessed by ANOVA followed by Student’s t-test using Bonferroni correction to account for multiple comparisons. *** marks p-values < 0.001, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Flow Cytometry

Journal: Scientific Reports

Article Title: UBXD1 is a mitochondrial recruitment factor for p97/VCP and promotes mitophagy

doi: 10.1038/s41598-018-30963-z

Figure Lengend Snippet: UBXD1 levels influence mitophagic flux. ( A ) Using CRISPR/Cas9, UBXD1 was targeted in HeLa cells and several alleles of UBXD1 were replaced with a reporter cassette coding for secreted Gaussia luciferase. Shown are schematics of CRISPR/Cas9 strategy, ( B ) PCR analysis of reporter integration, ( C ) and levels of UBXD1 in HeLa and HeLa UBXD1-low detected using mouse anti-UBXD1 antibodies. ( D ) HeLa cells HeLa UBXD1-low transfected with mKeima-T2A-Parkin-myc3 were treated for 12 hours with 10, 25 or 50 µM CCCP or left untreated as controls and were analyzed by flow cytometry. The box plot represents 5 independent experiments. ( E ) Shown are representative density plots of the analysis shown in D. Statistical significance was assessed by ANOVA followed by Student’s t-test using correction to account for multiple comparisons according to Holm. * marks p-value < 0.05, **p-value < 0.01, n.s. – no significant difference.

Article Snippet: To detect UBXD1 protein, protein lysates of HeLa cells were prepared using RIPA buffer, separated on 12%SDS-PAGE, and analyzed using mouse anti-UBXD1 (Abcam, 80659), mouse α-GAPDH (1:3000; Santa Cruz #sc-32233), anti-FLAG (1:1000, Sigma F1804), anti-myc (1:2000, Sigma M5546) and anti-mouse HRP (1:20000; ThermoFisher) antibodies.

Techniques: CRISPR, Luciferase, Transfection, Flow Cytometry

Journal: Nature protocols

Article Title: Easi -CRISPR protocol for creating knock-in and conditional knockout mouse models using long ssDNA donors

doi: 10.1038/nprot.2017.153

Figure Lengend Snippet: The procedure involves three broad stages: (i) assembling of CRISPR Ribonucleoprotein components (crRNA + tracrRNA + Cas9 Protein: ctRNP) and generating a long ssDNA donor (Steps 1–40); (ii) preparation of Easi-CRISPR components, their microinjection into mouse zygotes and generation of founder offspring (Steps 41–53), and; (iii) genotyping of offspring (Steps 54–59). All experimental procedures using animals should be carried out according to relevant institutional regulations for animal usage.

Article Snippet: REAGENTS FOR MOUSE GENOTYPING Cell Lysis Solution (Qiagen, cat. no. 158908) Protein Precipitate Solution (Qiagen, cat. no. 158912) DNA Hydration Solution (Qiagen, cat. no. 158914) TaKaRa Taq (TaKaRa, cat. no. R001) or PrimeSTAR HS DNA Polymerase (TaKaRa, cat. no. R010) 2xGC buffer (TaKaRa, cat. no. 9154) Allele-In-One Mouse Tail Direct Lysis Buffer (KURABO, cat. no. ABP-PP-MT01500) Agarose (Phenix Research Products, cat. no. RBA 500) 50X TAE Buffer (ThermoFisher Scientific, cat. no. BP1332–20) Nuclease free water (many common vendors) TA Cloning® Kit, with pCRTM2.1 Vector (Life Technologies, cat. no. K202020) LB agar Ampicillin-100, Plates (Sigma, cat.no.

Techniques: CRISPR, Microinjection

Journal: Nature protocols

Article Title: Easi -CRISPR protocol for creating knock-in and conditional knockout mouse models using long ssDNA donors

doi: 10.1038/nprot.2017.153

Figure Lengend Snippet: (a) Genotyping floxed alleles. Primer sets 1–2 and 3–4 amplify single LoxP insertion at the two separate sites but cannot suggest if they are inserted in cis or in trans. Correct insertion genotype (in cis) can be determined by PCR using the primer sets 5–4 and 1–6, and confirmed by sequencing the PCR products. Note that the 3’ ends of primers 5 & 6 bind to the first 15 bases of LoxP sites (primer 5; 5’-NNNNNNNNNNNNNNNNNNNNataacttcgtatagc-3’: primer 6; 5’-NNNNNNNNNNNNNNNNNNNNataacttcgtataat-3’). (b) Genotyping knock-in (and knock-down) alleles. Three PCRs are performed; one each for 5’ and 3’ junctional regions (primer sets 7–8 and 9–10), and the third PCR for insert-specific regions (primer set 12–11). PCR with outer primer sets (7–11 and 12–10) amplify longer PCR fragments, including the full knock-in cassette. If amplification of longer sequences is not successful (for example if primers 7–11 and 12–10 that amplify nearly the full length of the cassette do not work well), alternate primers within the insertion cassette should be tried. Similarly, alternate primers of 9 and 8 (within the cassette) can also be tried to obtain smaller amplicons (if larger products cannot be amplified efficiently for the junctional PCRs). The amplified fragments in both (a) and (b) should be sequenced to ensure sequence fidelity. The examples of PCRs of primer pairs 1–2 and 3–4 (for Pitx1 floxing), and 7–8 & 9–10 (MMP9-T2AmCitrine knock-in) were previously reported in Quadros et al., 2017[9]. All experimental procedures using mice was carried out according to Tokai University institutional regulations for animal usage (permit number: #165009).

Article Snippet: REAGENTS FOR MOUSE GENOTYPING Cell Lysis Solution (Qiagen, cat. no. 158908) Protein Precipitate Solution (Qiagen, cat. no. 158912) DNA Hydration Solution (Qiagen, cat. no. 158914) TaKaRa Taq (TaKaRa, cat. no. R001) or PrimeSTAR HS DNA Polymerase (TaKaRa, cat. no. R010) 2xGC buffer (TaKaRa, cat. no. 9154) Allele-In-One Mouse Tail Direct Lysis Buffer (KURABO, cat. no. ABP-PP-MT01500) Agarose (Phenix Research Products, cat. no. RBA 500) 50X TAE Buffer (ThermoFisher Scientific, cat. no. BP1332–20) Nuclease free water (many common vendors) TA Cloning® Kit, with pCRTM2.1 Vector (Life Technologies, cat. no. K202020) LB agar Ampicillin-100, Plates (Sigma, cat.no.

Techniques: Sequencing, Knock-In, Knockdown, Amplification

Journal: bioRxiv

Article Title: SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

doi: 10.1101/2021.02.01.429135

Figure Lengend Snippet: (A) Denaturing PAGE resulting from activity assays performed with the CARF protein TTHB144 and a 5’ Cy5 labelled reporter RNA. Activation of TTHB144 due to cOAs produced by the TtCmr complex was monitored by offering fully complementary (T) target RNAs, or target RNA with mismatches in segments one (S1), four (S4) or six (S6), or by a single mismatch in the CAR (C1). A fully non-target RNA (NT) was used as a control. (B) Limit of detection (LOD) assay using a synthetic SARS-CoV-2 E-gene and a fluorophore-quencher reporter RNA masking construct measured over time. A non-target RNA (NT) was used as a control. (C) Schematic overview of the 2-step reaction setup consisting out of (1) a (RT)-LAMP based pre-amplification step and (2) a T7-based in vitro transcription and type III CRISPR detection step. (D) Limit of detection assay using the 2-step setup (depicted in panel C), with a SARS-CoV-2 synthetic full RNA genome as target. (E) Detection of SARS-CoV-2 in human swab samples. Ct-values of qPCR analysis ( Orf1ab gene) of 81 samples are depicted on the X-axis with the true negative samples displayed as not determined (ND). See Table S2 for Ct-values of qPCR analysis of SARS-CoV-2 samples and respective Scope tool score. (F) One-pot LAMP-CRISPR limit of detection assay on a synthetic SARS-CoV-2 E-gene.

Article Snippet: The subsequent CRISPR detection was performed by adding 8.75 μL of CRISPR Mix (1X TtCmr Activity Assay Buffer, ~62.5 nM TtCmr complex, 500 nM TTHB144, 2 μL NTP Buffer Mix (NEB #E2050), 25 U Hi-T7 RNA polymerase (NEB #M0658), 250 nM RNaseAlert™ QC System v2 (ThermoFisher) and 8 mM final concentration MgCl).

Techniques: Activity Assay, Activation Assay, Produced, Construct, Amplification, In Vitro, CRISPR, Detection Assay

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Strategy to identify Repeat A-interacting proteins in vitro . (B) Proteins enriched by WT over ΔGC-core Repeat A ( p < 0.05, two-sided t-test, n=3). (C) GO terms of GC-core-specific proteins (top 20). (D) Relative recovery of SR proteins across Repeat A RNAs. Columns, independent experiments. (E) RMCE- Xist WT RIP-seq with antibodies against controls, SPEN, and SR and/or RNA-processing proteins. (F) RIP-seq signal within Repeat A relative to total Xist . Dots, independent experiments. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: In Vitro

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Promega Diamond-stained urea-PAGE gels of in-vitro-transcribed, internally biotinylated Repeat A RNAs used for biotinylated RNA pulldown experiments. (B) Representative silver-stained SDS-PAGE gel of proteins recovered by biotinylated Repeat A mutant pulldowns or no-RNA control pulldown. The remaining 85% of each sample was analyzed by quantitative proteomics. (C) Validation of quantitative proteomics results by biotinylated RNA pulldown and western blotting for the proteins RBM15, U2AF2, and SRSF1. (D) Quantification of the proportion of RIP-seq signal in within Repeat A relative to total reads per million. Dots, values from independent experiments.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Staining, In Vitro, SDS Page, Mutagenesis, Control, Quantitative Proteomics, Biomarker Discovery, Western Blot

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Genotyping PCR for independent clonal lines of RMCE- Xist WT Δ Spen cells or non-targeting gRNA-treated control cells generated by CRISPR-Cas9. PCR1 was performed using primers flanking either side of the deletion; PCR2 and PCR3 were performed using primers flanking the upstream and downstream Spen gRNA target sites. (B) Confirmation of expression of WT and mutant Halo-SPEN-V5 proteins by IP-WB or WB. (C) Xist silencing activity determined by RNA-seq, as in , for non-targeting control cells and Δ Spen cells with and without WT Halo-SPEN-V5 expression. Asterisks, significant differences by two-sided t-test. (D) Quantification of exon 1 aberrant splicing for each RMCE- Xist cell line (dots), as done in . Asterisks, values significantly different from WT by two-sided t-test. (E and F) Quantification of RS/SR and RE/ER dipeptide content in each IDR of SPEN by percentage of residues (E) or overall residue count (F). (G) Gene ontology analysis of the 75 proteins recovered with SPEN RRM1-4 two-fold more than with RRM2-4 in two independent experiments, showing the 10 most significantly enriched terms. (H) Gene ontology analysis of the 44 non-chromosome 1 (chr1) genes with expression significantly correlated with SPEN across tissue types in the TCGA Tumor dataset (FDR < 0.1). An additional 59 chr1 genes were significantly correlated with SPEN in this dataset but were omitted from this analysis because their correlations with SPEN , a gene on chr1, cannot be decoupled from cancer-associated copy-number variations.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Control, Generated, CRISPR, Expressing, Mutagenesis, Activity Assay, RNA Sequencing, Residue

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Xist abundance across genotypes (RNA-seq). Dots, independent clonal lines. (B) Halo-SPEN-V5-interacting proteins ranked by proportion of peptides recovered. (C) GO terms of SPEN-interacting proteins (top 10). (D) V5 IP, Halo and SRSF1 western blots (no-rescue vs. Halo-SPEN-V5 ESCs). (E) SPEN domains and deletion mutants. IDRs, MobiDB. (F) SPEN-Repeat A association for WT vs. mutants by input-normalized Halo RIP-qPCR. Dots, qPCR triplicates (≥2 independent experiments). Asterisks, significantly different from WT. Crosses, significantly different from RRM1-4. (G) Enrichment of proteins, by class, in RRM1-4 vs. RRM2-4 IP-MS/MS. “(0),” no recovery with RRM2-4. (H) Peptide counts of SR and/or speckle proteins in RRM1-4 or RRM2-4 IP-MS/MS. Dots, independent experiments. (I) Motifs from top 1500 non- Xist , non- Spen Halo RIP-seq peaks across genotypes. (J) Significant SPEN -correlated genes from GTEx (guilt-by-association; p < 0.01 and FDR < 0.1, see Methods). (K) Overlap of significant SPEN -correlated genes (FDR < 0.1) from the GTEx, TCGA Tumor (non-chr1), and TCGA Normal datasets. (L) GO terms of SPEN -correlated genes from GTEx (top 10 and 14 th ). Excepting (J), p -values, two-sided t-tests: (*/†), p < 0.05; (**), p < 0.01. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: RNA Sequencing, Western Blot, Protein-Protein interactions

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) O-MAP schematic; HRP, horseradish peroxidase. (B) Proteins differentially recovered by O-MAP from WT and Δ Spen cells ( p < 0.05, two-sided t-test, n=4). (C) Repeat A association (input-normalized RIP-qPCR) in non-targeting-control and Δ Spen ESCs derived from RMCE- Xist WT, and RMCE- Xist AWCG+ΔGG cells. Values relative to non-targeting controls. Dots, qPCR triplicates from ≥2 independent experiments. Values above non-targeting-control bars, experiment-averaged IP/input relative to IgG control. (D) RIP-qPCR as in (C), shown relative to IgG. (E) Xist abundance (RNA-seq) in RMCE- Xist WT ESCs treated with non-targeting control siRNA (siNT) and 1000, 25, or 10 ng/mL dox, or protein-targeting siRNAs and 1000 ng/mL dox. Values relative to siNT-1000. Dots, independent experiments. (F) Xist silencing activity as in , for siRNA-treated cells. (G) Median silencing activity vs. Xist abundance. Δ Spen data, , S5C. Dashed line, silencing expected for corresponding levels of Xist in siNT ESCs. P -values, two-sided t-tests relative to WT or non-targeting controls: (*) indicates p < 0.05; (**) indicates p < 0.01. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Control, Derivative Assay, RNA Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) SRSF1-Repeat A association in SM33 or RMCE- Xist ESCs by input-normalized RIP-qPCR. Srsf2 mRNA, control. Dots, qPCR triplicates from two independent experiments. (B) SPEN- vs. SRSF1-Repeat A association (RIP-qPCR). r , p : data fit to linear regression. (C) RBNQ with SRSF1 RRM1-2 and Repeat A. Dots, values from two independent experiments. Error bars, standard deviation of qPCR triplicates. (D) RBNQ across Repeat A RNAs using fixed SRSF1 RRM1-2 concentration. Dots, qPCR triplicates values, ≥2 independent experiments. (E) FKBP1A F36V -SRSF1 degradation by dTAG V -1 in cells derived from RMCE- Xist WT. (F) SPEN-Repeat A association in WT or FKBP1A F36V - Srsf1 cells by input-normalized RIP-qPCR. Dots, qPCR triplicates from independent clonal lines. (G) Xist abundance (RNA-seq) in FKBP1A F36V - Srsf1 cells, with/without dox and with/without dTAG V -1. Dots, independent lines, relative to dox(+), dTAG V -1(-) condition. (H) Xist silencing activity as in , for FKBP1A F36V - Srsf1 cells treated as in (G). (I) MCP-MS2 tethering to assess SPEN recruitment to Xist ΔRepA-XB-6x(U-spacer-3xMS2). (J) SPEN-6x(U-spacer-3xMS2) association across MCP genotypes by input-normalized RIP-qPCR. Dots, qPCR triplicates from ≥2 independent experiments. (K) Xist abundance (RNA-seq) across genotypes. Dots, independent experiments. (L) Xist silencing activity as in across MCP genotypes. “n.s.,” not significantly different. P -values, two-sided t-tests relative to WT or EGFP unless specified by black bars: (*), p < 0.05; (**), p < 0.01. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Control, Standard Deviation, Concentration Assay, Derivative Assay, RNA Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Levels of Repeat A recovery by input-normalized FLAG RIP-qPCR from RMCE- Xist WT cells expressing no FLAG-tagged construct and RMCE- Xist WT and ΔCCUGC cells expressing SRSF2-3xFLAG. Dots, qPCR triplicate values from one experiment. (B) Coomassie blue-stained SDS-PAGE gel of two independent preps of SBP-tagged SRSF1 RRM1-2 used for replicates of equilibrium RBNQ experiments. (C) Genotyping PCR for parental RMCE- Xist WT control cells or independent clonal lines of FKBP1A F36V - Srsf1 cells generated by CRISPR-Cas9 and homology-directed repair. PCR was performed using primers flanking either side of the FKBP1A F36V degron tag insertion. (D) Quantification of exon 1 aberrant splicing for each RMCE- Xist cell line (dots), as done in . Asterisks, values significantly different from WT by two-sided t-test. (E) Western blot confirmation of expression of untagged EGFP or various MCP-tagged proteins in RMCE- Xist ΔRepA-XB-6x(U-spacer-3xMS2) cells. (F) Confirmation of MCP-tagged protein tethering by input-normalized RIP-qPCR using antibodies against SRSF1 (left) or FLAG (right). Cells expressing untagged EGFP served as a negative control. Dots, qPCR triplicate values from two independent experiments. Asterisks, significantly different from untagged EGFP control by two-sided t-test. (G) Levels of SPEN association with WT, mutant, or synthetic Repeat A in RMCE- Xist WT, ΔU- spacer, or ΔGC-core cells or RMCE- Xist ΔRepA-XB-6x(U-spacer-3xMS2) cells, determined by input-normalized RIP-qPCR. Dots, qPCR triplicate values from two independent experiments (one that included ΔGC-core). Asterisk, significantly different from WT by two-sided t-test (two-sided one-sample t-test for ΔGC-core). (H and I) Plots of Xist RNA abundance as a function of SPEN-Repeat A association level (H) or SRSF1-Repeat A association level (I). Values of r and p reflect fit to a linear regression models with the indicated data. The ss234 deletion reduces Xist abundance independent of Repeat A and was thus omitted.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Expressing, Construct, Staining, SDS Page, Control, Generated, CRISPR, Western Blot, Negative Control, Mutagenesis

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Repeats 6 and 7 depicted according to structural model from Lu et al. , SPEN is recruited to Repeat A predominantly by proteins that bind motifs in the GC-rich core, whereas the U-rich spacers play minor roles. SPEN and GC-rich-core-bound proteins enable association with factors involved in transcriptional elongation and RNA processing. (B) Sensing of SR protein-rich transcriptional hubs by Repeat A and SPEN.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques:

Journal: Nature Communications

Article Title: Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

doi: 10.1038/s41467-017-01732-9

Figure Lengend Snippet: Cas9:tracrRNA complexed with RNA or hybrid crXNAs are nuclease competent. a Design of the crRNA, crDNA and crHyb sequence for GFP target site 1 (GFP cr1). DNA shown in uppercase black, RNA shown in lowercase red. b Cas9, tracrRNA and crXNA molecules were incubated with linear double-stranded DNA harbouring the GFP target sequence. Reaction products were analysed by agarose gel electrophoresis. The expected undigested and digested fragments are exemplified on the right. c Semiquantitative analysis of the cleavage of the linear DNA fragment by crDNA (blue)-, crHyb (black)- and crRNA (red)-guided Cas9:tracrRNA complexes with three independent guide sequences. Bars show mean + SD, n = 3–7

Article Snippet: For qPCR detection of template depletion, Cas9 cleavage assays were conducted using 5, 2.5, 1.25 or 0.625 nM Cas9 (New England Biolabs), 15 nM tracrXNA (Dharmacon and Eurogentec), 15 nM crXNA molecules (IDT and Eurogentec), 1× Cas9 reaction buffer (New England Biolabs), 0.75 nM eGFP and AAVS1 target DNA.

Techniques: Sequencing, Incubation, Agarose Gel Electrophoresis

Journal: Nature Communications

Article Title: Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

doi: 10.1038/s41467-017-01732-9

Figure Lengend Snippet: RNA-rich hybrid crXNA molecules can direct specific Cas9:tracrRNA nuclease activity in cells. Quantification of insertion and deletion (InDel) events at the AAVS1 locus 5 days after electroporation of Cas9:tracrHyb.v2:crXNA complexes. Dotted line show threshold determined from crDNA samples

Article Snippet: For qPCR detection of template depletion, Cas9 cleavage assays were conducted using 5, 2.5, 1.25 or 0.625 nM Cas9 (New England Biolabs), 15 nM tracrXNA (Dharmacon and Eurogentec), 15 nM crXNA molecules (IDT and Eurogentec), 1× Cas9 reaction buffer (New England Biolabs), 0.75 nM eGFP and AAVS1 target DNA.

Techniques: Activity Assay, Electroporation

Journal: Nature Communications

Article Title: Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

doi: 10.1038/s41467-017-01732-9

Figure Lengend Snippet: Cas9:tracrRNA can function with a crXNA composed of 88% DNA and crHyb show enhanced specificity. a RNA nucleosides within the crHyb for GFP target 1 were sequentially converted to DNA and activity assessed by DNA fragmentation assay. Nucleotides coloured as in Fig. . b A single base C > A mismatch was introduced into crRNA and crHyb molecules and activity assessed. All bars show mean + SD, n = 3–13. c Kinetics of template degradation driven by Cas9:tracrRNA complexed with crHyb (black) or crRNA (red), either with perfect complementary (left) or with a single base-pair mismatch (right). Graphs show normalised template abundance (%) over time, with Cas9 protein at 5 nM (solid line), 2.5 nM (long dash line), 1.25 nM (dot dash line) or 0.625 nM (dotted line). Error bars show SD of 3–4 independent measurements

Article Snippet: For qPCR detection of template depletion, Cas9 cleavage assays were conducted using 5, 2.5, 1.25 or 0.625 nM Cas9 (New England Biolabs), 15 nM tracrXNA (Dharmacon and Eurogentec), 15 nM crXNA molecules (IDT and Eurogentec), 1× Cas9 reaction buffer (New England Biolabs), 0.75 nM eGFP and AAVS1 target DNA.

Techniques: Activity Assay, DNA Fragmentation Assay

Journal: Nature Communications

Article Title: Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

doi: 10.1038/s41467-017-01732-9

Figure Lengend Snippet: DNA in the crXNA molecule affects target-binding affinities. a Nucleoside composition of AAVS1 crXNA molecules; RNA shown in lowercase red, DNA in uppercase black. b BLI sensogram showing association kinetics to immobilised AAVS1 target DNA of Cas9:tracrRNA complexed with crRNA (red), crHyb (black), crDNA (blue), crR.Hyb (green) or crD.Hyb (purple). Shaded areas show SD of three independent measurements. c SwitchSENSE sensogram showing association phase of Cas9:tracrRNA:crXNA complexes binding to target DNA. Shaded areas show SD of four independent measurements

Article Snippet: For qPCR detection of template depletion, Cas9 cleavage assays were conducted using 5, 2.5, 1.25 or 0.625 nM Cas9 (New England Biolabs), 15 nM tracrXNA (Dharmacon and Eurogentec), 15 nM crXNA molecules (IDT and Eurogentec), 1× Cas9 reaction buffer (New England Biolabs), 0.75 nM eGFP and AAVS1 target DNA.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

doi: 10.1038/s41467-017-01732-9

Figure Lengend Snippet: Biochemical and cellular characterisation of tracrHyb. a Design of the split-tracrRNA molecule. b Quantification of in vitro cleavage of target DNA by Cas9 complexes with crRNA (red) or crHyb (black) and combinations of single or split tracr molecules. c Design of the single tracr hybrid molecule (tracrHyb.2). d SwitchSENSE traces showing association phase of Cas9:tracrHyb.2:crXNA complexes binding to target DNA, coloured as in 3b. e Quantification of InDel events at the AAVS1 locus 5 days after electroporation of complexes composed of Cas9 and crRNA (red), crR.Hyb (green) or crDNA (black), and tracrRNA or tracrHyb.2. Dotted line shows threshold determined from crDNA samples and. For all charts, bars show mean + SD, n > 3

Article Snippet: For qPCR detection of template depletion, Cas9 cleavage assays were conducted using 5, 2.5, 1.25 or 0.625 nM Cas9 (New England Biolabs), 15 nM tracrXNA (Dharmacon and Eurogentec), 15 nM crXNA molecules (IDT and Eurogentec), 1× Cas9 reaction buffer (New England Biolabs), 0.75 nM eGFP and AAVS1 target DNA.

Techniques: In Vitro, Binding Assay, Electroporation