MA5-13449

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of SNRPB was achieved by transfecting HeLa cells with SNRPB specific siRNAs (Silencer® select Product # s13220 and s13221). Western blot analysis (Fig a) was performed using Modified whole cell extract from the SNRPB knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-SNRPB Mouse Monoclonal Antibody (Product # MA5-13449, 0.2 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to SNRPB.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), NTERA-2 (Lane 2), NIH/3T3 (Lane 3), Hep G2 (Lane 4), K-562 (Lane 5), SH-SY5Y (Lane 6), HEK-293 (Lane 7) and A-431 (Lane 8). The blots were probed with Mouse Anti-SNRPB Monoclonal Antibody (Product # MA5-13449, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 24 kDa and 30 kDa band corresponding to SNRPB was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), NTERA-2 (Lane 2), NIH/3T3 (Lane 3), Hep G2 (Lane 4), K-562 (Lane 5), SH-SY5Y (Lane 6), HEK-293 (Lane 7) and A-431 (Lane 8). The blots were probed with Mouse Anti-SNRPB Monoclonal Antibody (Product # MA5-13449, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 24 kDa and 30 kDa band corresponding to SNRPB was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SNRPB was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with SNRPB Mouse monoclonal antibody (Product # MA5-13449) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of SNRPB was achieved by transfecting HeLa cells with SNRPB specific siRNA (Silencer® select Cat # s13220, s13221). Immunofluorescence analysis was performed on HeLa cells (untransfected, panel a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with SNRPB specific siRNA (panel c,f) Cells were fixed, permeabilized, and labelled with SNRPB Mouse monoclonal Antibody (Product # MA5-13449, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Reduction of specific signal was observed upon siRNA mediated knockdown (panel c,f) confirming specificity of the antibody to SNRPB(green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Detection of binding of endogenous SNRPB protein to specific RNA using Anti-SNRPB Antibody: RNA Immunoprecipitation (RIP) was performed using Anti-SNRPB Mouse Monoclonal Antibody (Product # MA5-13449, 3 µg) on clarified whole cell lysate from 4 million HCT 116 cells. Normal Rabbit IgG was used as a negative IP control. Immunoprecipitated RNA was purified by RiboPure™ RNA Purification Kit (Product # AM1924) and analyzed by RT-PCR using the Power SYBR® Green RNA-to-CT™ 1-Step Kit (Product # 4389986) with primer pairs over U1 snRNA (positive) and 18S rRNA, ACTB mRNA, GAPDH mRNA (negative). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Immunohistochemistry of kidneys demonstrating the overexpression of ribonucleoproteins induced by heavy metal salts. Note that at the beginning of the trial (control), the expression is faint or absent, whereas at the end of the intoxication trial, ribonucleoprotein expression notably increases.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 RECTAS directly interacts with CDC-like kinase 1 (CLK1), and it enhances cellular SRSF6 activity. a Structures of RECTAS and b-RECTAS. b Extent of IKBKAP -FD exon 20 inclusion represented by GFP/RFP ratio, following treatment with biotin (50 uM), RECTAS (2 uM), b-RECTAS (2, 10, and 50 uM), or solvent only (0.1% DMSO) for 24 h in reporter-transfected HeLa cells. c , d Western blotting of pull-down products by b-RECTAS (0, 100, or 200 nmol/reaction) for HeLa cells transfected with a flag-tagged CLK1 expression vector ( c ) or GST-tagged recombinant CLK1 ( d ). RECTAS (500 nmol) was incubated with 100 nmol b-RECTAS for the competition assay in ( d ). e , f Western blot ( e ) for phosphor SRSF6 in FD patient primary fibroblasts (P1 and P2), treated with RECTAS (10 uM) or solvent only (0.1% DMSO) for 24 h. f Phosphor SRSF6 in FD fibroblasts (P1) was quantified at 0, 6, 10, 24 h after RECTAS (10 uM) treatment (columns, 0 h, 6 h, 10 h, and 24 h), as well as 10 h after washout of compound following RECTAS treatment (10 uM) for 24 h (column, wo). Lamin B1 served as a loading control in ( e ). g Western blot for U1-70k and SmB/B' in pull-down products with biotin-conjugated RNA surrounding IKBKAP exon 20 donor site with (oAM154) or without (oAM153) IVS20 + 6 T > C mutation (labeled as FD and WT, respectively). Pull-down was conducted in the presence of 10 uM RECTAS with 1% DMSO, or 1% DMSO only. RNA (-), control sample without RNA bait. Input, HeLa nuclear extract of 5% input am

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. Identification and characterization of truncated U12 snRNAs in human cells. ( A ) Effects of mutations in the terminal base-pair of U12 snRNA stem-loop III on full-length U12 and U12 fragment levels. Top: Plasmid constructs for expressing tag-U12 with various combinations of nucleotides in the 84 and 150 positions were transfected into HeLa cells. Co-transfected F30-2xdBroccoli expression plasmid served as a transfection control. Total RNA from transfected HeLa cells was analyzed by northern blot with U12, U11, tag-U12 and Broccoli probes. Bottom: Quantification of full-length U12 (blue) and U12 fragment (orange) levels. Band intensities were normalized by the sum of all band intensities of each replicate, and the mean full-length U12 snRNA signal of WT tag-U12 (C-G) was then set to 1. Statistical significance was determined using one-way ANOVA followed by Dunnett's test. Significance levels above each bar refer to comparisons between WT tag-U12 and the indicated variant. Based on pair-wise comparisons (ANOVA with Tukey's test), differences in fragment levels between the group of variants showing low levels of tag-U12 fragments (C-G, G-C, G-G) and the group with high levels of fragments (U-G, U-A, C-C, U-C, C-A) were all statistically significant (*-***). In contrast, none of the pair-wise comparisons within the two groups showed statistically significant differences in fragment levels. ( B ) Location of truncation sites of U12 snRNA fragments determined by Sanger s

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. Different sensitivities of MTAP -deleted and MTAP-rescued cells to Type I PRMT inhibition. ( A-D ) MTAP -deleted cells were reconstituted with the stable re-expression of MTAP. These isogenic cell pairs were cultured for 8 days in the presence of DMSO or 1 muM of MS023 (0.1 muM for MiaPaCa2 cells). Different cell types display differing degrees of sensitivity to MS023, which we established empirically. The cell growth curves are plotted for A549 (A), MiaPaCa2 (B), A172 (C) and MCF-7 (D) cell lines. The expression levels of MTAP are shown for each pair of cell lines. ( E ) A scatter plot showing the sensitivities of MTAP -deleted cells to PRMT5 knockdown. Each dot in the scatter plot represents a cancer cell line. Data retrieved from the DepMap project ( https://depmap.org/portal/ ). ( F ) Using total cell lysates from MTAP -deleted and MTAP-rescued cells, the levels of SDMA, ADMA, SmB methylation (Y12) and MTAP were analyzed by western blot.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. Disruption of the Spliceosome following Tau Expression in Drosophila (A and B) Pan-neuronal human Tau R406W disrupts expression of multiple spliceosomal proteins in 1-day-old adult fly head homogenates. Western blots (A) were probed for Tau or spliceosome proteins and normalized to the loading control, GAPDH (n = 4 replicates for quantification, B). The Y12 antibody recognizes both SmB and SmD3. (C) mRNA expression was also examined in 1-day-old adult heads (n = 3). (D and E) Whole-mount stains of 1-day-old adult fly brains (D) reveal depletion of SmB/D3 and U1A/SNF protein (red, n = 15 for quantification, E). Nuclei are colabeled with 4',6-diamidino-2-phenylindole (DAPI; grayscale). Scale bar: 20 mum. (F) Glial expression of Tau WT ( repo-GAL4 ) induces cytoplasmic foci (arrowheads) of SmB/D3 (Y12, red) that colocalize with phospho-Tau aggregates (green) in 10-day-old adult brains. Nuclei are labeled with DAPI (blue). Boxed region is magnified at right. Quantification (n > 9) reveals 14.86% +- 2.3% of phospho-Tau aggregates colabeling for Sm proteins; aggregates were not observed in controls. Scale bar: 10 mum. See also Figure S3A . All error bars denote mean +- SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. Loss of SmB , Encoding a Core Spliceosome Factor, Causes Reduced Survival and Progressive Locomotor Impairment (A) The SmB locus, including single coding exon (CDS, yellow), untranslated regions (UTRs, gray), and transposable elements. To generate SmB MG , recombination-mediated cassette exchange was performed using SmB MI , introducing a coding exon for GFP, flanked by flexible linkers (Ls) and splice acceptor and donor sequences (SA/SDs). The deficiency strain (red), Df(2L)BSC453 , deletes ~51 kb including the entire SmB locus. The transgenic genomic rescue strain, SmB GR , carries a ~90-kb bacterial artificial chromosome (BAC, CH321-75P02, green), including SmB. See also Table S2 . (B) SmB MG/MG homozygotes demonstrate expression of the GFP-SmB fusion protein at levels comparable with controls (n = 3 for quantification, SmB protein normalized to Actin). (C) GFP-SmB (green) is localized to the nucleus (DAPI, grayscale) in brains from SmB MG/MG adults. Scale bar: 20 mum. Boxed region (top) is magnified below. (D) SmB MG/MG adults (black) exhibit reduced survival and this phenotype is partially rescued by the SmB genomic rescue (blue) (n > 313 per genotype). See also Figure S4A . (E) SmB MG/MG adults also manifest progressive locomotor impairment (n > 5 groups). See also Figure S4B . (F and G) Both the SmB MG/MG survival (F, n > 288) and locomotor (G, n > 4 groups) phenotypes were rescued by pan-neuronal expression of wild-type SmB . All error bars denote mean +- SE