Journal: BioEssays : news and reviews in molecular, cellular and developmental biology

Article Title: Cytoplasmic mRNPs revisited: Singletons and condensates.

doi: 10.1002/bies.202000097

Figure Lengend Snippet: FIGURE 1 Schematic overview of the different cytoplasmic mRNP particles present in eukaryotic cells and their interrelationships. Protein exchange or remodeling steps are indicated between the different mRNP states: pre-translational mRNPs, active translating mRNPs, stress granules and P-bodies. (a) Mature messenger RNAs coated with RNA-binding proteins exit the nucleus through the nuclear pore. At this stage, three characteristic RNA-binding complexes are present in pre-translational mRNPs; namely CBP20/80 (forming the cap-binding complex CBC), the exon-junction complex (EJC), and PABPN1 (together with PABPC1). In the cytoplasm, pre-translational mRNPs are transported in a

Article Snippet: Antibodies against CBP80 (E-7, Santa Cruz Biotechnology), YBX1 (ab12148, Abcam) and PABPC1 (HPA067156, Atlas Antibodies) were directly conjugated to Alexa Fluor 488, 568 and 647 dyes, respectively.

Techniques: RNA Binding Assay, Binding Assay

Journal: BioEssays : news and reviews in molecular, cellular and developmental biology

Article Title: Cytoplasmic mRNPs revisited: Singletons and condensates.

doi: 10.1002/bies.202000097

Figure Lengend Snippet: FIGURE 2 Super-resolution fluorescence microscopy images acquired by Structured Illumination Microscopy of pre-translational mRNPs, P-bodies and stress granules in HeLa cells. Upper panel—Direct immunofluorescent staining of a linear pre-translational mRNP unit with three of its protein constituents: CBP80 (green), YBX1 (red), and PABPC1 (cyan). CBP80 is at the 5′ terminus of the mRNP (5′-cap), YBX1 is coating the mRNA body, and PABPC1 is binding to the poly(A) tail at the 3′ terminus. Antibodies against CBP80 (E-7, Santa Cruz Biotechnology), YBX1 (ab12148, Abcam) and PABPC1 (HPA067156, Atlas Antibodies) were directly conjugated to Alexa Fluor 488, 568 and 647 dyes, respectively. Scale bars = 5 μm (whole cell image) and 0.1 μm (zoomed-in image). Lower panels—Image of a whole cell with its P-bodies visualized by the presence of DCP1a-GFP and a zoomed-in view of one P-body (left). Indirect immunofluorescence staining of G3BP in a cell stressed with 1 mM sodium arsenite for 20 minutes showing stress granules in the cytoplasm and a detailed view of two stress granules (right). A mouse monoclonal anti-G3BP (ab56574, Abcam) primary antibody and a secondary antibody anti-mouse Alexa Fluor 488 were used. Scale bars = 5 μm (whole cell images) and 0.5 μm (zoomed-in images).

Article Snippet: Antibodies against CBP80 (E-7, Santa Cruz Biotechnology), YBX1 (ab12148, Abcam) and PABPC1 (HPA067156, Atlas Antibodies) were directly conjugated to Alexa Fluor 488, 568 and 647 dyes, respectively.

Techniques: Fluorescence, Microscopy, Staining, Binding Assay, Immunofluorescence

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Inhibition of tRNA charging activates the ISR without inducing RNP granules. ( A ) U-2 OS cells were treated with 0.2% DMSO carrier, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) for 1 h, and Western blotting for phosphorylated and total eIF2α was done. Total protein ( n = 3 independent replicates) and molecular weights (in kilodaltons) are shown. ( B ) Translation activity was measured using bioorthogonal noncanonical amino acid tagging in cells treated as in A . Methionine or azidohomoalanine (AHA) was added 10 min prior to collection. The ratio of nascent to total protein is shown from n = 3 independent replicates. ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A and fixed, and nuclei were stained with Hoechst (blue). The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥305 cells were counted in each condition. Scale bars, 10 µm. ( D ) Cells were treated as in C , and immunofluorescence and fluorescence in situ hybridization were done to detect UBAP2L (magenta), PABPC1 (cyan), and poly(A) RNA with oligo(dT) probes (yellow) ( n = 2 independent experiments). ( E ) Cells were treated with 0.2% DMSO, 20 µM HF plus 0.2% DMSO, 250 µM arsenite, or 1.25 µM thapsigargin for 1 h, and the percentage of cells with SGs was quantified ( n = 3 independent experiments); ≥287 cells were counted per treatment. ( F ) Cells were treated, and immunofluorescence microscopy was done as in D to detect the P-body marker DCP1A. Quantification of the percentage of cells with P-bodies (PBs) from n = 3 independent experiments is shown; ≥364 cells were counted per treatment. ( G ) Cells were treated as in C to detect XRN1 (magenta) and EDC4 (yellow) by immunofluorescence microscopy ( n = 2 independent experiments). ( H ) U-2 OS cells were treated as in E , and immunofluorescence microscopy was done to detect DCP1A. The percentage of cells with PBs from n = 3 independent experiments is shown; ≥351 cells were counted per treatment across all replicates. Representative images are shown with the average ± SEM for each experiment, and green, gray, and pink points represent the average of each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: PABPC1 , Proteintech , 66809 , 1:100 (immunofluorescence).

Techniques: Inhibition, Western Blot, Activity Assay, Stable Transfection, Expressing, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Microscopy, Marker

Journal: Stem Cell Research & Therapy

Article Title: Deer thymosin beta 10 functions as a novel factor for angiogenesis and chondrogenesis during antler growth and regeneration

doi: 10.1186/s13287-018-0917-y

Figure Lengend Snippet: Antibodies

Article Snippet: Vascular endothelial growth factor-D (VEGFD) , Rabbit , Polyclonal antibody , Elabscience, China , E-AB-33213 , WB: 1 μg/ml (1:1000).

Techniques: Control, Labeling

Journal: The Journal of biological chemistry

Article Title: A complex with poly(A)-binding protein and EWS facilitates the transcriptional function of oncogenic ETS transcription factors in prostate cells.

doi: 10.1016/j.jbc.2023.105453

Figure Lengend Snippet: Figure 2. PABPC1 is an interacting partner of ERG and ctEWS. A, immunoblot of PABPC1 in various prostate cell lines. B, immunoprecipitation with anti- ERG antibody, immunoblotted for PABPC1. C, immunoblot of affinity pulldown experiments of purified GST-tagged EWS fragments or PABPC1 using purified His-ERG bound to magnetic beads. D, immunoprecipitation from VCaP lysate with anti-EWS, immunoblotted for PABPC1 (top) and pulldown of purified PABPC1 by either purified ctEWS or ntEWS bound to magnetic beads (bottom). E, immunoblot of an affinity pulldown of PABPC1 using purified oncogenic ETS factors (ERG, ETV1, ETV4, and ETV5) and the nononcogenic FLI1. Purified protein quantity was measured by Ponceau staining (lower). F, immuno- precipitation of FLAG-tagged ETV4 from RWPE cells, immunoblotted for endogenous PABPC1. G, immunoblot of coimmunoprecipitation of endogenous ERG and PABPC1 in nuclear and cytoplasmic fractions of RWPE-ERG cells. H, immunoblot of coimmunoprecipitation of hemagglutinin-tagged ctEWS in nuclear and cytoplasmic fractions of PC3-ctEWS cells. I, immunoblot of PABPC1 in nuclear and cytoplasmic fractions of RWPE-empty vector or RWPE-ERG cells. H3 and GAPDH are nuclear (N) and cytoplasmic (C) markers, respectively. J, diagrams of ERG truncations show locations of pointed (PNT) and ETS DNA- binding domains (ETS). K, immunoblot of affinity pulldown of PABPC1 from PC3 nuclear extracts using the purified ERG fragments shown in (J) bound to magnetic beads. ERG 392 to 455 only is also fused to maltose-binding protein (MBP). Purified ERG protein quantity was visualized by Ponceau staining. ctEWS, C terminus of EWS; GST, glutathione-S-transferase; PABPC1, poly-adenylate–binding protein.

Article Snippet: CoraLite 594–conjugated PABPC1 antibody (Proteintech Cat# CL594-66809; 1:200) in PBS containing 0.1% Tween-20 and 1% BSA were added overnight at 4 C in a humidified chamber.

Techniques: Western Blot, Immunoprecipitation, Magnetic Beads, Staining, Plasmid Preparation, Binding Assay

Journal: The Journal of biological chemistry

Article Title: A complex with poly(A)-binding protein and EWS facilitates the transcriptional function of oncogenic ETS transcription factors in prostate cells.

doi: 10.1016/j.jbc.2023.105453

Figure Lengend Snippet: Figure 3. PABPC1, ERG, and EWS form a complex. A, coomassie blue staining of purified GST-ctEWS, His-ERG, and His-PABPC1. B, anti-GST immunoblot of pulldown of GST-ctEWS (all lanes) with nickel beads in the absence or presence of His-ERG or His-PABPC1. C, anti-ERG Immunoblot of pulldown of His-ERG using glutathione beads in the presence or absence of GST-ctEWS, and with or without increasing concentrations of His-PABPC1. D, immunoblot of PABPC1 or hemagglutinin-ctEWS from VCaP cells–expressing hemagglutinin-ctEWS and shRNAs-targeting luciferase (−) or PABPC1 (+). Pull down is binding to nickel beads coated with purified His-ERG. Densitometry values normalized to VCaP-ctEWS plus luciferase-targeted shRNA are displayed. E, a replicate using same knockdown cells as shown in (D), but including loading control for ERG (Ponceau), and immunoblot for endogenous full-length EWS. F, densitometry quantification of ctEWS in pulldown from (D) and (E) plus an additional biological replicate (mean + SEM). ctEWS, C terminus of EWS; GST, glutathione-S- transferase; PABPC1, poly-adenylate–binding protein.

Article Snippet: CoraLite 594–conjugated PABPC1 antibody (Proteintech Cat# CL594-66809; 1:200) in PBS containing 0.1% Tween-20 and 1% BSA were added overnight at 4 C in a humidified chamber.

Techniques: Staining, Western Blot, Expressing, Luciferase, Binding Assay, shRNA, Knockdown, Control

Journal: The Journal of biological chemistry

Article Title: A complex with poly(A)-binding protein and EWS facilitates the transcriptional function of oncogenic ETS transcription factors in prostate cells.

doi: 10.1016/j.jbc.2023.105453

Figure Lengend Snippet: Figure 4. ERG promotes PABPC1 binding to chromatin. A, overlap of PABPC1 ChIP-seq peaks in RWPE-empty vector and RWPE-ERG cells. B, most overrepresented motifs in PABPC1 bound sites. C, most overrepresented biological process ontologies of nearest neighbor genes to PABPC1 ChIP-seq called peaks in RWPE-ERG cells using ChIP-ENRICH. D and E, metagene plot of PABPC1 ChIP reads from RWPE-empty vector and RWPE-ERG cells compared to ERG and EWS ChIP reads centered on called ERG (E) or EWS (F) peaks. ChIP, chromatin immunoprecitation; PABPC1, poly-adenylate–binding protein.

Article Snippet: CoraLite 594–conjugated PABPC1 antibody (Proteintech Cat# CL594-66809; 1:200) in PBS containing 0.1% Tween-20 and 1% BSA were added overnight at 4 C in a humidified chamber.

Techniques: Binding Assay, ChIP-sequencing, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: A complex with poly(A)-binding protein and EWS facilitates the transcriptional function of oncogenic ETS transcription factors in prostate cells.

doi: 10.1016/j.jbc.2023.105453

Figure Lengend Snippet: Figure 5. PABPC1 is important for biological functions of ERG in prostate cells. A, immunoblot of PABPC1 in RWPE cells–expressing ERG and shRNAs- targeting luciferase or PABPC1. Densitometry was performed to calculate the knockdown efficiency for each shRNA. Densitometry values normalized to RWPE-empty vector plus luciferase-targeted shRNA are displayed. B, trans-well migration assay of RWPE-empty vector and RWPE-ERG cells with PABPC1 or control luciferase targeted shRNAs. Migration was normalized to RWPE-empty vector cells with luciferase-targeted shRNA. C, trans-well migration assay of RWPE-KRAS (RWPE2) cells with PABPC1-targeted shRNA normalized to RWPE-KRAS plus luciferase-targeted shRNA. Immunoblot demonstrates PABPC1 knockdown. D, trans-well migration assay of PC3-ERG cells with PABPC1 shRNA normalized to PC3-ERG plus luciferase shRNA. E, relative number of colonies formed after low-density plating of VCaP cells with or without PABPC1 knockdown, shLuciferase is set to 1, n = 6. Immunoblot demonstrates PABPC1 knockdown. F, relative number of colonies formed following low-density plating of cell lines from (B). Colony number was normalized to RWPE-empty vector plus luciferase-targeted shRNA. G, relative number of colonies formed following low-density plating of cell lines from (B) with transient trans- fection of myristoylated AKT (mAKT). Colony number was normalized to RWPE-empty vector plus luciferase-targeted shRNA. H, luciferase assay of RWPE- empty vector and RWPE-ERG cells with or without PABPC1-targeted shRNAs. ERG activity was determined using a reporter containing seven consecutive repeats of the ETS-binding site GGAA. The mean and SEM of four biological replicates are plotted. I, RT-qPCR of three ERG target genes used in (39). Quantity of mRNA transcripts were first normalized to 18S, and then normalized to RWPE-ERG + shLuciferase. Unless otherwise indicated, all experiments are plotted as the mean and SEM of three biological replicates. p-values were calculated using Student’s t test, where * indicates a p-value < 0.05, ** indicates a p-value < 0.01, and *** indicates a p-value < 0.001. GST, glutathione-S-transferase; PABPC1, poly-adenylate–binding protein; qPCR, quantitative PCR.

Article Snippet: CoraLite 594–conjugated PABPC1 antibody (Proteintech Cat# CL594-66809; 1:200) in PBS containing 0.1% Tween-20 and 1% BSA were added overnight at 4 C in a humidified chamber.

Techniques: Western Blot, Expressing, Luciferase, Knockdown, shRNA, Plasmid Preparation, Migration, Control, Activity Assay, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: EMBO Reports

Article Title: Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase

doi: 10.1038/s44319-025-00397-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-PABP (Boster) was used at 1:10,000.

Techniques: SYBR Green Assay, Imaging, Recombinant, Sequencing, Software, Microscopy, Ion-Mobility Spectrometry, Mass Spectrometry