rabbit anti-son nbp1–88706  (Novus Biologicals)

Journal: Science Advances

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1126/sciadv.adp7727

Figure Lengend Snippet: ( A ) Scheme of ARTR-seq: Scaffold protein is immunostained by primary and secondary antibodies (Abs) sequentially. pAG-RTase then binds to the antibody to initiate reverse transcription in situ. The generated biotinylated cDNAs are collected and prepared for sequencing. ( B ) Representative image showing colocalization of Alexa Fluor 647 (AF647)–labeled pAG-RTase (magenta), Alexa Fluor 568 (AF568)–labeled secondary antibody against anti-SON primary antibody (blue), and biotinylated cDNA detected by Alexa Fluor 488 (AF488)–labeled antibody against biotin (green). The biotinylated cDNA signal in the image without the use of primary antibody (-Pri-Ab) is also shown at a fivefold lower contrast. Dashed line marks the nucleus boundary. ( C ) Two-dimensional (2D) histogram showing the correlation between I NSE determined through targeting SON and SRRM2 (in log 2 scale) in HeLa cells. Genes with lfcSE < 1 from DESeq analysis of ARTR-seq are included. Linear function ( y = ax + b ) is used to fit I NSE,SRRM2 to I NSE,SON . ( D ) Genome tracks showing ARTR-seq reads generated from targeting SON or SRRM2 and from samples without primary antibody (-Pri-Ab), mapped to MALAT1 gene. Additional genome track for -Pri-Ab is shown using autoscale for the number of reads. ( E ) RNA FISH images showing LAMA5 and P4HB transcript. RNA FISH probes were labeled with AF647 (magenta). Nuclear speckles were immunostained with AF488 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (gray). The zoomed-in image shows one nucleus in each case. ( F ) Correlation between speckle partition coefficient ( R NS/cell ) measured by RNA FISH imaging and I NSE values determined by ARTR-seq. R NS/cell was calculated as the ratio of fluorescence signal inside nuclear speckles to that in the entire cell, which should theoretically correspond to I NSE . In (C) and (F), “ N ” reports the number of genes included in the analysis, and “ R ” reports Pearson’s correlation coefficient.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus, RRID:AB_11006030), mouse anti-SRRM2 antibody (1:200 dilution, Sigma-Aldrich, RRID:AB_477511), or mouse anti-SRSF1 antibody (1:250 dilution, Invitrogen, RRID:AB_2533080) for 1 hour at room temperature followed by three-times wash with 1× PBS.

Techniques: Reverse Transcription, In Situ, Generated, Sequencing, Labeling, Staining, Imaging, Fluorescence

Journal: Science Advances

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1126/sciadv.adp7727

Figure Lengend Snippet: ( A ) Scatter plot showing randomly selected genes from group A, B, and C genes and corresponding I NSE(exon) under NT and Plad B treatment conditions in HeLa cells. ( B ) Genome tracks showing poly (A) + RNA-seq (pink) and nuclear RNA-seq (blue) under NT and Plad B treatment conditions for selected genes: THOC6 (group A), TUBB4B (group B), and NCL (group C). Selected efficiently excised or inefficiently excised introns are highlighted in cyan and red boxes, respectively. ( C ) Schematic description of the RT-PCR assay. After reverse transcription of extracted total RNA, primers located on two adjacent exons of selected introns were used for amplification, and the PCR products were analyzed by electrophoresis. ( D ) Representative immunofluorescence images showing nuclear speckles upon SON / SRRM2 double knockdown (KD) and treated with control siRNA (siC). SON (green) and SRRM2 (magenta) were stained with AF488 and CF568 respectively. Nuclei were stained with DAPI (gray). ( E ) Histogram of SON and SRRM2 fluorescence intensity distribution under KD and siC treatment. ( F ) Violin plot showing the number of speckles per cell for KD and siC treatment. Nuclear speckles were identified by a user-defined threshold of the total intensity of the SON and SRRM2 signals, applied to both knockdown and control samples. ( G ) Representative electrophoresis analysis of RT-PCR products from THOC6 , TUBB4B , and NCL upon KD and siC treatment. ( H ) Apparent unexcised fractions of selected introns were calculated by ratios of the intensity of the unexcised band and the sum of the unexcised band and excised band. Each bar reports the mean unexcised fraction from two biological replicates. “ N ” reports the total number of cells included in each dataset in (E) and (F). P values calculated with unpaired t tests are reported above each violin plot or bar in (F) and (H).

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus, RRID:AB_11006030), mouse anti-SRRM2 antibody (1:200 dilution, Sigma-Aldrich, RRID:AB_477511), or mouse anti-SRSF1 antibody (1:250 dilution, Invitrogen, RRID:AB_2533080) for 1 hour at room temperature followed by three-times wash with 1× PBS.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Electrophoresis, Immunofluorescence, Knockdown, Control, Staining, Fluorescence

Journal: Science Advances

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1126/sciadv.adp7727

Figure Lengend Snippet: ( A ) IRFinder analysis showing heat shock–induced up-regulation of intron retention. The number of intron retention events with more than 15% increase (∆IR >15% ) or decrease (∆IR <-15% ) are labeled. ( B ) Percentage of group A genes and group B and C genes without and with taking the subset of genes containing ∆IR >15% introns. P value: Fisher’s exact test. ( C ) Violin plot showing the group A–like sequence feature associated with three groups of introns (ΔIR >15% , ΔIR (−15%, 15%) , ΔIR <-15% ). The GC content, intron length, splice site strength, and intronic ML score are compared for three groups of introns. ( D ) Viability of HeLa cells under nuclear speckle disruption via SON/SRRM2 double KD or under siC upon heat shock stress or NT. Hoechst staining reflects the total cell population, whereas trypan blue stains dead cells. Cell viability was calculated as one minus the fraction of dead cells (1 − N Dead / N Total ), where N Dead and N Total are the number of dead cells and total cell number, respectively. P values calculated with unpaired t test are reported above each violin plot and box plot. Error bars report SD from three biological replicates (in black dots). ( E ) Immunofluorescence image of nuclear speckles under NT and heat shock conditions. ( F ) Violin plot showing the speckle size in heat shock compared to NT. ( G ) 2D histogram showing the correlation between I NSE(exon) values under heat shock and NT. “ N ” reports the number of introns in (A) and (C), the number of genes in (B) and (G), and the number of speckles in (F).

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus, RRID:AB_11006030), mouse anti-SRRM2 antibody (1:200 dilution, Sigma-Aldrich, RRID:AB_477511), or mouse anti-SRSF1 antibody (1:250 dilution, Invitrogen, RRID:AB_2533080) for 1 hour at room temperature followed by three-times wash with 1× PBS.

Techniques: Labeling, Sequencing, Disruption, Staining, Immunofluorescence

Journal: iScience

Article Title: RNA molecules display distinctive organization at nuclear speckles

doi: 10.1016/j.isci.2024.109603

Figure Lengend Snippet: Effect of SRSF1 and hnRNPA1 knockdown on the intra-speckle organization of RNAs containing SRSF1 motifs in exon and hnRNPA1 motifs in intron (A) Schematic illustration of MUT S1-H1 . (B) Immunofluorescence (IF) quantification of each cell chosen for imaging treated with scramble siRNA, siRNA against SRSF1 and siRNA against hnRNPA1. Immunofluorescence is quantified by staining SRSF1 and hnRNPA1 proteins with their respective antibodies and imaging with a 750 nm laser under scramble and knockdown conditions and computing the average intensity of the whole cell. (C) Representative SMLM images of MUT S1-H1 treated with scramble siRNA, siRNA against SRSF1 and siRNA against hnRNPA1. FISH signals corresponding to hnRNPA1 and SRSF1 motifs in the RNAs are shown in magenta and green respectively. Immunostaining of scaffold protein SON is shown in blue. Scale bars represent 5 μm (white) and 1 μm (black). (D and F) Population distribution of SRSF1 and hnRNPA1 motif signals as a function of the normalized distance from the center (D), and from the edge (F) of the speckle for MUT S1-H1 under knockdown conditions. (E and G) Population-weighted mean normalized distance of SRSF1 and hnRNPA1 signal from the center (E), and from the edge (G) for each speckle for MUT S1-H1 under knockdown conditions. Error bars in the population vs. distance plots report the standard deviation from two replicates, each replicate containing at least 48–72 nuclear speckles collected from 4 to 6 cells. Scatterplots are generated by combining all nuclear speckles (96–144) from two replicates. Values in scatterplot represent mean ± SEM. p values in the scatterplots are calculated with paired sample Wilcoxon signed rank test (black, one-sided) and two sample t test (magenta and green, one-sided), with ∗ p < 5e-2, ∗∗ p < 1e-2, ∗∗∗ p < 1e-3. Replicates are biological replicates collected from different dishes of cells and measured on different days. Also see Figures S11 and .

Article Snippet: Solutions of primary antibodies were prepared in blocking solution using the following dilutions: mouse antibody against SRRM2 (1:2000, Sigma Aldrich, #S4045), mouse antibody against SRSF1 (1:250, Invitrogen, #32–4600), rabbit antibody against hnRNPA1 (1:100, Abcam, #ab177152), rabbit antibody against SON (1:200, Invitrogen, #PA5-54814).

Techniques: Immunofluorescence, Imaging, Staining, Immunostaining, Standard Deviation, Generated

Journal: iScience

Article Title: RNA molecules display distinctive organization at nuclear speckles

doi: 10.1016/j.isci.2024.109603

Figure Lengend Snippet:

Article Snippet: Solutions of primary antibodies were prepared in blocking solution using the following dilutions: mouse antibody against SRRM2 (1:2000, Sigma Aldrich, #S4045), mouse antibody against SRSF1 (1:250, Invitrogen, #32–4600), rabbit antibody against hnRNPA1 (1:100, Abcam, #ab177152), rabbit antibody against SON (1:200, Invitrogen, #PA5-54814).

Techniques: Recombinant, Modification, Electron Microscopy, RNA Extraction, Software, Imaging