rabbit polyclonal anti pspc1  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Luciferase activity in A549 cells transfected with siRNAs targeting paraspeckle components. Data are represented as mean ± SD (n = 3). siNEAT1_1 and 2 indicate two different siRNAs both targeting the whole NEAT1.The data for RBM14, RBMX, RBM5, HNRNPA1, HNRNPA2B1, HNRNPK and PSPC1 were reproduced from Figure S4C. (B) Quantitative PCR analysis of viral RNA species of fragment NP normalised to GAPDH in A549 cells with knockdowns of paraspeckle proteins, infected with WSN (MOI 3) at 6hpi. Data are presented as log-fold-change of as mean ± SD relative to the siNT condition (n=3). (C) Schematic of NONO knockout and rescue workflow. CRISPR-Cas9 with two sgRNAs generated NONO KO1 and KO2 A549 cell lines. Lentiviral overexpression of mEGFP-NONO or mEGFP (control) in KO cells was used for rescue. Wild-type, NONO KO, and rescued cells were analysed for paraspeckle function and viral replication. Created in BioRender. https://BioRender.com/t41h404 . (D) Luciferase activity in wt, NONO KO1, and KO2 in A549 cells (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (E) Luciferase activity in wt, NONO KO1, and KO2 A549 cells lentivirally overexpressing mEGFP (control) or mEGFP-NONO (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (F) Density plot of log2-fold-changes of proteins cross-linked to NONO, NP and NS1 identified by AP-MS (NONO infected vs. NONO mock). Proteins cross-linked to NP and NS1 (orange) are co-depleted in infected cells compared to proteins not linked to NP or NS1 (grey). (G) Log2-fold changes of interactors identified by AP-MS against NONO (infected vs. mock) and NP (infected vs. infected isotype control). Proteins cross-linked to NONO (pink), NP (orange), and non-associated proteins (grey) are shown. (H) Selected protein categories enriched in both NP and NONO-mock AP-MS datasets from G (see also Table S3). (I) Schematic representation of paraspeckles disruption: IAV proteins, particularly NP and NS1, disrupt paraspeckle integrity by binding core proteins like SFPQ and NONO, and possibly NEAT1, initiating paraspeckle disassembly. As the infection progresses, PA-X promotes NEAT1 degradation, while POL II inhibition further destabilises paraspeckles, leading to their complete disruption. Created in BioRender. https://BioRender.com/y54j344 . (A-B, D-E) Data are represented as mean ± SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Infection, Knock-Out, CRISPR, Generated, Over Expression, Control, Disruption, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Cross-linking network between paraspeckle and viral proteins. Pink – essential for paraspeckle formation, blue – important, yellow – localised to paraspeckles but dispensable , gray – other proteins cross-linked to both paraspeckle and orange - viral proteins. (B) Paraspeckle structure, showing the interaction between NEAT1 long noncoding RNA and proteins such as SFPQ and NONO (left) and the NEAT1 isoforms, NEAT1_1 and NEAT1_2, and the position of FISH probes/qPCR primers on NEAT1 used in this study (right). Created in bioRender. https://BioRender.com/b92y974 (C) AP-MS of NONO versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). NONO and its known interactors are coloured pink and viral proteins - orange. (D) AP-MS of NP versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). Proteins that were also identified in SHVIP are highlighted. Proteins coloured as in E. (E) Maximum projection of confocal microscopy images of A549 cells infected with WSN (MOI 3) at 4, 8, and 12 hpi. NEAT1 - magenta, vRNA (PB2 fragment) - green, and DNA (DAPI) - grey. (F) CV of NEAT1_2 (NEAT1_1 in case of MEF) in the nucleus across different cell lines infected with WSN. (G) Number of paraspeckles per nucleus across different cell lines infected with WSN. (F-G) Data are represented as mean + SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Infection, Confocal Microscopy

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Luciferase activity in A549 cells transfected with siRNAs targeting paraspeckle components. Data are represented as mean ± SD (n = 3). siNEAT1_1 and 2 indicate two different siRNAs both targeting the whole NEAT1.The data for RBM14, RBMX, RBM5, HNRNPA1, HNRNPA2B1, HNRNPK and PSPC1 were reproduced from Figure S4C. (B) Quantitative PCR analysis of viral RNA species of fragment NP normalised to GAPDH in A549 cells with knockdowns of paraspeckle proteins, infected with WSN (MOI 3) at 6hpi. Data are presented as log-fold-change of as mean ± SD relative to the siNT condition (n=3). (C) Schematic of NONO knockout and rescue workflow. CRISPR-Cas9 with two sgRNAs generated NONO KO1 and KO2 A549 cell lines. Lentiviral overexpression of mEGFP-NONO or mEGFP (control) in KO cells was used for rescue. Wild-type, NONO KO, and rescued cells were analysed for paraspeckle function and viral replication. Created in BioRender. https://BioRender.com/t41h404 . (D) Luciferase activity in wt, NONO KO1, and KO2 in A549 cells (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (E) Luciferase activity in wt, NONO KO1, and KO2 A549 cells lentivirally overexpressing mEGFP (control) or mEGFP-NONO (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (F) Density plot of log2-fold-changes of proteins cross-linked to NONO, NP and NS1 identified by AP-MS (NONO infected vs. NONO mock). Proteins cross-linked to NP and NS1 (orange) are co-depleted in infected cells compared to proteins not linked to NP or NS1 (grey). (G) Log2-fold changes of interactors identified by AP-MS against NONO (infected vs. mock) and NP (infected vs. infected isotype control). Proteins cross-linked to NONO (pink), NP (orange), and non-associated proteins (grey) are shown. (H) Selected protein categories enriched in both NP and NONO-mock AP-MS datasets from G (see also Table S3). (I) Schematic representation of paraspeckles disruption: IAV proteins, particularly NP and NS1, disrupt paraspeckle integrity by binding core proteins like SFPQ and NONO, and possibly NEAT1, initiating paraspeckle disassembly. As the infection progresses, PA-X promotes NEAT1 degradation, while POL II inhibition further destabilises paraspeckles, leading to their complete disruption. Created in BioRender. https://BioRender.com/y54j344 . (A-B, D-E) Data are represented as mean ± SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Infection, Knock-Out, CRISPR, Generated, Over Expression, Control, Disruption, Binding Assay, Inhibition

Journal: eLife

Article Title: Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia

doi: 10.7554/elife.69162

Figure Lengend Snippet: Figure 4. Paraspeckle proteins p54nrb and PSCP1, but not SFPQ, are ITAFs of the FGF1 IRES. (A) Schema of paraspeckle and DBHS proteins. (B–D) FGF1 IRES activity upon knock-down of SFPQ (B), P54nrb (C) or PSPC1 (D) in HL-1 cell (Figure 4—figure supplement 1—source data 1) transduced with Lucky Luke bicistronic reporter during normoxia or hypoxia was measured as in Figure 2. Cells were harvested 72 hr after siRNA treatment. The IRES activity values have been normalized to the control siRNA. Histograms correspond to means ± standard

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- P54nrb (rabbit polyclonal) Santacruz Sc- 67016 Dilution 1:200 (capillary Western) Dilution 1:400 (classical Western) Antibody Anti- PSPC1 (rabbit polyclonal) bethyl laboratory A303- 205A Dilution 1:100 (capillary Western) Dilution 1:1000 (classical Western) Antibody Anti- SFPQ (mouse monoclonal) Abcam Ab11825 Dilution 1:100 Antibody Anti- FGF1 (rabbit polyclonal) Abcam Ab207321 Dilution 1:25 Antibody Anti- nucleolin (rabbit polyclonal) Novus biological NB600- 241 Dilution 1:50 Antibody Anti- Histone H3 (rabbit polyclonal) Cell Signaling 4499 Dilution 1 : 10000 Antibody Anti- GAPDH (mouse monoclonal) SantaCruz Sc- 32233 Dilution 1:1000 Antibody Mouse total IgG (mouse polyclonal) Sigma I5381 2 mg/mL Antibody Anti- eIF2α (rabbit polyclonal) Cell Signaling Technology 9721 Dilution 1:50 Antibody Anti- phospho- eIF2α (mouse monoclonal) Cell Signaling Technology 2103 Dilution 1:50 Antibody Anti- p21 (mouse monoclonal) Santacruz Sc- 6246 Dilution 1:50 Antibody Anti- HA (mouse monoclonal) Sigma H9558/H3663 2.4 mg/mL (72 μg) Antibody Anti- rabbit- peroxidase conjugate (donkey polyclonal) Jackson ImmunoResearch 711- 035- 152 Dilution 1:10000 Antibody Anti- mouse- peroxidase conjugate (rabbit polyclonal) Jackson ImmunoResearch 715- 035- 150 Dilution 1:10000 Antibody Rabbit detection module Protein Simple DM- 001 10 μl Antibody Mouse detection module Protein Simple DM- 002 10 μl Strain, strain background (Escherichia coli) Top10 InVitrogen C404003 Godet, Roussel et al. eLife 2022;11:e69162.

Techniques: Activity Assay, Knockdown, Transduction, Control

Journal: eLife

Article Title: Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia

doi: 10.7554/elife.69162

Figure Lengend Snippet: Figure 9. Model of IRESome formation in the paraspeckle. According to the present data, we propose that the paraspeckle may be a recruitment platform for IRES-containing mRNAs in hypoxic cardiomyocytes. Neat1 and proteins present in the paraspeckle (among them major paraspeckle components such as p54nrb and PSPC1) would assemble the IRESome, then mRNA would be exported from the nucleus and translated in the cytosol. Identification of Neat1 in the cytoplasm suggests that it might be part of the IRESome and have a direct role in translation. However this latter hypothesis remains to be elucidated.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- P54nrb (rabbit polyclonal) Santacruz Sc- 67016 Dilution 1:200 (capillary Western) Dilution 1:400 (classical Western) Antibody Anti- PSPC1 (rabbit polyclonal) bethyl laboratory A303- 205A Dilution 1:100 (capillary Western) Dilution 1:1000 (classical Western) Antibody Anti- SFPQ (mouse monoclonal) Abcam Ab11825 Dilution 1:100 Antibody Anti- FGF1 (rabbit polyclonal) Abcam Ab207321 Dilution 1:25 Antibody Anti- nucleolin (rabbit polyclonal) Novus biological NB600- 241 Dilution 1:50 Antibody Anti- Histone H3 (rabbit polyclonal) Cell Signaling 4499 Dilution 1 : 10000 Antibody Anti- GAPDH (mouse monoclonal) SantaCruz Sc- 32233 Dilution 1:1000 Antibody Mouse total IgG (mouse polyclonal) Sigma I5381 2 mg/mL Antibody Anti- eIF2α (rabbit polyclonal) Cell Signaling Technology 9721 Dilution 1:50 Antibody Anti- phospho- eIF2α (mouse monoclonal) Cell Signaling Technology 2103 Dilution 1:50 Antibody Anti- p21 (mouse monoclonal) Santacruz Sc- 6246 Dilution 1:50 Antibody Anti- HA (mouse monoclonal) Sigma H9558/H3663 2.4 mg/mL (72 μg) Antibody Anti- rabbit- peroxidase conjugate (donkey polyclonal) Jackson ImmunoResearch 711- 035- 152 Dilution 1:10000 Antibody Anti- mouse- peroxidase conjugate (rabbit polyclonal) Jackson ImmunoResearch 715- 035- 150 Dilution 1:10000 Antibody Rabbit detection module Protein Simple DM- 001 10 μl Antibody Mouse detection module Protein Simple DM- 002 10 μl Strain, strain background (Escherichia coli) Top10 InVitrogen C404003 Godet, Roussel et al. eLife 2022;11:e69162.

Techniques:

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet: NEAT1_2 Recruits TDP-43 into Paraspeckles (A) A schematic overview of the mRNA-interactome analysis strategy used for identifying relocalized RBPs upon paraspeckle formation. Peptides were identified by UV crosslinking and capturing mRNA-RBP complexes using Oligod(T)25-bound magnetic beads followed by mass spectrometry. Because deletion of the NEAT1 pA site promotes paraspeckle formation in undifferentiated hESCs ( Figure 1 ), comparing peptide counts in NEAT1ΔpA with WT cells detects resulting changes in mRNA-RBP occupancy. Further details regarding global mRNA-interactome analysis are given in D–S2F. (B) A Volcano plot displaying fold changes of mRNA-bound RBPs in NEAT1ΔpA undifferentiated hESCs and their respective statistical score (t test, n = 3 biological replicates per condition). Previously identified paraspeckle proteins are labeled red. (C) Western blot analysis of TDP-43 and non-paraspeckle control RBPs of the lysate versus the supernatant fraction after mRNA depletion by Oligod(T) capture, from UV-crosslinked WT and NEAT1ΔpA undifferentiated hESCs. Right: quantification (t test, n = 3, ∗∗ p < 0.01). (D) The positions of TDP-43 cross-linked sites (red bars) in NEAT1 that were identified by TDP-43 iCLIP (from Tollervey et al., 2011 ). The 1∼2-kb deleted regions (black boxes) in the NEAT1ΔUG cell line, and the positions of the three longest stretches of UG repeats within the transcript (yellow bars) are indicated. (E–G) Representative maximum projection photomicrographs from RNA-FISH and immunofluorescence of NEAT1_2 and the paraspeckle markers PSPC1 (E) and TDP-43 (F) in WT and NEAT1ΔUG HAP-1 cells. Blue, DAPI (nuclear stain). Scale bars, 10 μm. (G) shows quantification of the TDP-43 immunofluorescence signal in DAPI and NEAT1 segmented areas corresponding to nuclei and paraspeckles, respectively. More than 200 cells were analyzed per group; Mann-Whitney U test, ∗∗∗ p < 0.0001. The threshold was set on a ratio of TDP-43/ NEAT1 signal as described in the . (H) Diagram of NEAT1 corresponding to (C), with the position of an ectopic stretch of 60 UG repeats. (I and J) Representative maximum projection photomicrographs from NEAT1 RNA-FISH and TDP-43 immunofluorescence (I) and analysis of co-localization (J). Cell lines included WT, NEAT1ΔUG , and NEAT1ΔUG+UG60 HAP1 cells. Cell numbers and statistical analysis were as in (F). Scale bars, 10 μm.

Article Snippet: The coverslips were subsequently washed with TBST (1 × TBS containing 0.1% Tween 20) and incubated with 1 × blocking solution (Blocking reagent [Roche] diluted with TBST) for blocking at RT for 1 h. Then, the coverslips were incubated with primary antibodies, Anti-Digoxigenin mouse monoclonal antibody (clone 21H8, Abcam), Anti-TDP-43 rabbit polyclonal antibody (Proteintech, 1:100), anti-PSPC1 rabbit polyclonal antibody (1:1000; ) in 1 × blocking solution at room temperature for 1 h, washed three times with TBST for 5 min, and incubated with secondary antibodies, anti-mouse IgG Alexa 488, and anti-rabbit IgG, Alexa-568 (both Thermo Fisher Scientific]) in 1 × blocking solution at RT for 30 min, and washed three times with TBST for 5 min.

Techniques: Magnetic Beads, Mass Spectrometry, Labeling, Western Blot, Control, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet:

Article Snippet: The coverslips were subsequently washed with TBST (1 × TBS containing 0.1% Tween 20) and incubated with 1 × blocking solution (Blocking reagent [Roche] diluted with TBST) for blocking at RT for 1 h. Then, the coverslips were incubated with primary antibodies, Anti-Digoxigenin mouse monoclonal antibody (clone 21H8, Abcam), Anti-TDP-43 rabbit polyclonal antibody (Proteintech, 1:100), anti-PSPC1 rabbit polyclonal antibody (1:1000; ) in 1 × blocking solution at room temperature for 1 h, washed three times with TBST for 5 min, and incubated with secondary antibodies, anti-mouse IgG Alexa 488, and anti-rabbit IgG, Alexa-568 (both Thermo Fisher Scientific]) in 1 × blocking solution at RT for 30 min, and washed three times with TBST for 5 min.

Techniques: Recombinant, Produced, Gentle, Knock-Out, Reverse Transcription, TaqMan Assay, Labeling, Staining, Flow Cytometry, Western Blot, Clarification Assay, Membrane, Sample Prep, Sequencing, Derivative Assay, Next-Generation Sequencing, Gene Expression, Fractionation, Activation Assay, Nucleic Acid Electrophoresis, CRISPR, Plasmid Preparation, Modification, shRNA, Software

Journal: Cell

Article Title: CARM1 and Paraspeckles Regulate Pre-implantation Mouse Embryo Development

doi: 10.1016/j.cell.2018.11.027

Figure Lengend Snippet:

Article Snippet: rabbit polyclonal anti-PSPC1 , Santa Cruz Biotechnology , Cat# sc-84577; RRID: AB_2171459.

Techniques: Virus, Recombinant, Modification, In Vitro, Embryo Culture, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, Isolation, SYBR Green Assay, Negative Control, Software

Journal: EBioMedicine

Article Title: CPSF6 is a Clinically Relevant Breast Cancer Vulnerability Target

doi: 10.1016/j.ebiom.2017.06.023

Figure Lengend Snippet: Paraspeckles protein components are associated with poor outcome in breast cancer. A. Immunoblot blot analysis of total cell lysates of breast cancer cells using antibodies against SFPQ, P54nrb, PSPC1 and GAPDH. B. Confocal images of NEAT1 RNA–FISH (red) with nuclear DAPI counterstain (blue) in MCF7 and MDA-MB-231 cells. Results are expressed as mean ± SEM of triplicate of two independent experiments. **p < 0.01. Scale bars, 10. C–E. SFPQ, P54nrb and PSPC1 and mRNA levels in 144 normal and 1556 invasive breast cancer cases using Curtis dataset of ONCOMINE database. F–H. SFPQ, P54nrb and PSPC1 mRNA expression levels in 61 normal and 389 invasive breast cancer cases using TCGA dataset of ONCOMINE database. I–K. Kaplan-Meier survival curves of SFPQ, P54nrb and PSPC1 mRNA levels in association with patient outcome using RFS as an end point in breast cancer patients (3554, 1660 and 3554 respectively) (KM-plotter database). L–N. Kaplan-Meier survival curves SFPQ, P54nrb and PSPC1 mRNA levels in association with patient outcome using DMFS as an end point in breast cancer patients (1609, 664 and 1609 respectively) (KM-plotter database).

Article Snippet: Antibodies used were: anti-CPSF6 rabbit monoclonal antibody (abcam #ab175237), anti-Nudt21 mouse monoclonal antibody (Santa-Cruz #sc-81109), anti-SFPQ rabbit polyclonal antibody (abcam#ab38148) anti-P54nrb rabbit polyclonal antibody (Santa-Cruz # sc-67016), anti-PSPC1 rabbit polyclonal antibody (Santa-Cruz # sc-84576), anti-ADAR1 rabbit polyclonal antibody (abcam #ab126755), and anti-GAPDH mouse polyclonal antibody (Santa-Cruz # sc-365062).

Techniques: Western Blot, Expressing

Journal: EBioMedicine

Article Title: CPSF6 is a Clinically Relevant Breast Cancer Vulnerability Target

doi: 10.1016/j.ebiom.2017.06.023

Figure Lengend Snippet: CPSF6/Paraspeckles/ADAR1 protein complex in aggressive breast cancer. A. MDA-MB-231 cells were lysed and immunoprecipitations using a rabbit monoclonal antibody against CPSF6 or control normal rabbit IGg were performed. Western blotting was carried out using a polyclonal antibody against SFPQ (upper panel). Membrane was reprobed using a monoclonal antibody against CPSF6 (lower panel). TL: total cell lysates. B. MDA-MB-231 cells were lysed and immunoprecipitations using a rabbit polyclonal antibody against PSPC1 or control normal rabbit IGg were performed. Western blotting was carried out using a monoclonal antibody against CPSF6 (upper panel). Membrane was reprobed using a polyclonal antibody against PSPC1 (lower panel). TL: total cell lysates. C. Summary of CFIm and paraspeckles proteins physical interactions in MDA-MB-231 cells. D. CPSF6 immunoprecipitates of MDA-MB-231 and MCF7 cells were subjected to RT-qPCR using NEAT1 primers. Results are expressed as mean ± SEM of triplicates of two independent experiments. **p < 0.01. E. MDA-MB-231 and MCF7 cells were lysed and immunoprecipitations using a monoclonal antibody against CPSF6 or control normal rabbit IGg were performed. Western blotting was carried out using polyclonal antibody against ADAR1 (upper panel). Membrane was reprobed using a monoclonal antibody against CPSF6 (lower panel).

Article Snippet: Antibodies used were: anti-CPSF6 rabbit monoclonal antibody (abcam #ab175237), anti-Nudt21 mouse monoclonal antibody (Santa-Cruz #sc-81109), anti-SFPQ rabbit polyclonal antibody (abcam#ab38148) anti-P54nrb rabbit polyclonal antibody (Santa-Cruz # sc-67016), anti-PSPC1 rabbit polyclonal antibody (Santa-Cruz # sc-84576), anti-ADAR1 rabbit polyclonal antibody (abcam #ab126755), and anti-GAPDH mouse polyclonal antibody (Santa-Cruz # sc-365062).

Techniques: Control, Western Blot, Membrane, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: CPSF6 is a Clinically Relevant Breast Cancer Vulnerability Target

doi: 10.1016/j.ebiom.2017.06.023

Figure Lengend Snippet: CPSF6 is essential for maintaining physical integrity of CFIm, paraspeckles and ADAR1. A. Confocal immunofluorescence images (left panel) and immunoblot analysis (right panel) of MDA-MB-231-Scr and MDA-MB-231-Sh1-CPSF6 immunodetected using antibodies to CPSF6 and Nudt21. Quantification of western blots was performed for three independent experiments and expressed as mean ± SEM. ***p < 0.001. Scale bars, 10 μm. B. Confocal images of NEAT1 RNA–FISH (red) with nuclear DAPI counterstain (blue) in MDA-MB-231-Scr and MDA-MB-231-Sh1-CPSF6 cells. Scale bars, 10 μm. C. Immunoblot analysis of MDA-MB-231-Scr and MDA-MB-231-Sh1-CPSF6 using antibodies against SFPQ, p54nrb, PSPC1 and GAPDH (control). Right panels represent quantification of protein expression levels normalized by the control. Results are expressed as mean ± SEM of three independent experiments. **p < 0.01. D. Confocal immunofluorescence images (right panel) and immunoblot analysis (left panel) of MDA-MB-231-Scr and MDA-MB-231-Sh1-CPSF6 using antibody against ADAR1. Quantifications of western blots were performed for three independent experiments normalized by the control (GAPDH) and results are expressed as mean ± SEM. **p < 0.01, ***p < 0.01. Scale bars, 10 μm. E. Confocal immunofluorescence images (upper panel) and immunoblot analyses (lower panel) of MCF7-Scr and MCF7-Sh1-CPSF6 using antibodies against CPSF6, Nudt21, PSPC1, ADAR1 and GAPDH. Quantifications of western blots were performed for three independent experiments normalized by the control and results are expressed as mean ± SEM. **p < 0.01 and ns: not significant. Scale bars, 10 μm.

Article Snippet: Antibodies used were: anti-CPSF6 rabbit monoclonal antibody (abcam #ab175237), anti-Nudt21 mouse monoclonal antibody (Santa-Cruz #sc-81109), anti-SFPQ rabbit polyclonal antibody (abcam#ab38148) anti-P54nrb rabbit polyclonal antibody (Santa-Cruz # sc-67016), anti-PSPC1 rabbit polyclonal antibody (Santa-Cruz # sc-84576), anti-ADAR1 rabbit polyclonal antibody (abcam #ab126755), and anti-GAPDH mouse polyclonal antibody (Santa-Cruz # sc-365062).

Techniques: Immunofluorescence, Western Blot, Control, Expressing