bd mouse srsf1 12929 2 ap  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging

doi: 10.3390/ijms23031509

Figure Lengend Snippet: SRSF1 resists GC apoptosis and follicular atresia. ( A ) Venn diagram of the DEGs (differentially expressed genes) between HFs and AFs and miR-9820-5p target genes predicted by PITA, Miranda, and TargetSpy. ( B ) Differentially expressed SRSF1 in HFs and AFs detected by qRT-PCR. ( C , D ) Effective knockdown of SRSF1 by si-SRSF1 qRT-PCR and western blotting (WB). ( E ) The shift of GC apoptosis rates after SRSF1 knockdown was detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used in this study were polyclonal anti-SRSF1 (1:1000 dilution, 12929-2-AP, Proteintech Group, Rosemont, IL, USA), polyclonal anti-tubulin (1:1000 dilution, #6181S, Cell Signaling Technology, Boston, MA, USA), polyclonal anti-Cleaved Caspase-3 (C-CASP3) (1:1000 dilution, #9664S, Cell Signaling Technology, Boston, MA, USA), and the secondary antibody (1:2000 dilution, SA00E1–2, Proteintech Group, Rosemont, IL, USA).

Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging

doi: 10.3390/ijms23031509

Figure Lengend Snippet: miR-9820-5p promotes pGCs apoptosis by targeting SRSF1 . ( A , B ) The binding of miR-9820-5p and SRSF1-WT was predicted by RNAhybrid and verified by dual-luciferase activity analysis. ( C , D ) SRSF1 mRNA and protein levels after transfection of miR-9820-5p mimics. ( E , F ) SRSF1 mRNA and protein levels after transfection of miR-9820-5p inhibitors. ( G ) The shift of GC apoptosis rates after co-transfection of miR-9820-5p inhibitors and si-SRSF1 detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used in this study were polyclonal anti-SRSF1 (1:1000 dilution, 12929-2-AP, Proteintech Group, Rosemont, IL, USA), polyclonal anti-tubulin (1:1000 dilution, #6181S, Cell Signaling Technology, Boston, MA, USA), polyclonal anti-Cleaved Caspase-3 (C-CASP3) (1:1000 dilution, #9664S, Cell Signaling Technology, Boston, MA, USA), and the secondary antibody (1:2000 dilution, SA00E1–2, Proteintech Group, Rosemont, IL, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Cotransfection, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging

doi: 10.3390/ijms23031509

Figure Lengend Snippet: circSLC41A1 inhibits GCs apoptosis by upregulating SRSF1 through sponging miR-9820-5p. ( A ) The shift of SRSF1 mRNA levels after co-transfection of si-circSLC41A1 and miR-9820-5p inhibitors detected by qRT-PCR. ( B ) The shift of SRSF1 protein levels after co-transfection of the si-circSLC41A1 and miR-9820-5p inhibitors detected by WB. ( C ) Change of GC apoptosis rates after co-transfection of si-circSLC41A1 and miR-9820-5p inhibitors detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used in this study were polyclonal anti-SRSF1 (1:1000 dilution, 12929-2-AP, Proteintech Group, Rosemont, IL, USA), polyclonal anti-tubulin (1:1000 dilution, #6181S, Cell Signaling Technology, Boston, MA, USA), polyclonal anti-Cleaved Caspase-3 (C-CASP3) (1:1000 dilution, #9664S, Cell Signaling Technology, Boston, MA, USA), and the secondary antibody (1:2000 dilution, SA00E1–2, Proteintech Group, Rosemont, IL, USA).

Techniques: Cotransfection, Quantitative RT-PCR, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging

doi: 10.3390/ijms23031509

Figure Lengend Snippet: Schematic diagram of circSLC41A1 functions in GC apoptosis. circSLC41A1 can function by competing with SRSF1 mRNA for miR-9820-5p interactions, thereby alleviating the post-transcriptional repression of SRSF1 and thus resisting GC apoptosis and follicular atresia. circSLC41A1 also had the potential to encode small circSLC41A1-134aa peptides.

Article Snippet: The antibodies used in this study were polyclonal anti-SRSF1 (1:1000 dilution, 12929-2-AP, Proteintech Group, Rosemont, IL, USA), polyclonal anti-tubulin (1:1000 dilution, #6181S, Cell Signaling Technology, Boston, MA, USA), polyclonal anti-Cleaved Caspase-3 (C-CASP3) (1:1000 dilution, #9664S, Cell Signaling Technology, Boston, MA, USA), and the secondary antibody (1:2000 dilution, SA00E1–2, Proteintech Group, Rosemont, IL, USA).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A Hybridization chain reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1‐FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1‐CGG RNA foci co‐localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm. B Co‐localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit). C Co‐immunoprecipitation of indicated SRSF proteins with PP7‐tagged control sequence (partial nLUC sequence) to correct for non‐specific binding, AUG, and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7‐tagged CGG reporter. Source data are available online for this figure.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Hybridization, Immunocytochemistry, Immunoprecipitation, Sequencing, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A Schematic of (CGG)90‐EGFP construct and experimental outline for rough eye phenotype screening. B Quantitation of GMR‐GAL4‐driven uas‐(CGG)90‐EGFP eye phenotype with candidate modifiers ( n ≥ 30 flies/genotype). siNTC = siRNA against a non‐targeting control gene (mCherry). Different siRNA lines for the same target gene are numbered (#1 and #2). Error bars represent mean ± SD. C Representative photographs of fly eyes expressing either GMR‐GAL4 driver alone or with uas‐(CGG)90‐EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions (insertion). D Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n = 20–32/genotype. Error bars represent mean ± SD. E Representative photographs of fly eyes expressing GMR‐GAL4‐driven (GGGGCC)28‐EGFP with indicated uas‐siRNAs against fly SRSF genes in comparison with non‐targeting control (NTC) siRNA against LUC/luciferase. F Quantitation of (GGGGCC)28‐EGFP rough eye phenotype with SRSF modifiers ( n ≥ 30 flies/genotype). Error bars represent mean ± SD. G Representative photographs of fly eyes expressing GMR‐GAL4‐driven (GGGGCC)28‐EGFP at 29°C along with the quantifications of necrosis and eye width. n = 28–30/genotype. Error bars represent mean ± SD. H, I Survival assays of flies expressing (CGG)90‐EGFP under Tub5‐GS (H) and ELAV‐GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90‐EGFP was initiated with addition of drug starting 1 day post‐eclosion and continued through experiment (Log‐rank Mantel–Cox test; n = 98–101/genotype for Tub‐GS and n = 120–141/genotype for ELAV‐GS flies); * P < 0.05, ** P < 0.01. J Survival assays of (GGGGCC)28‐EGFP expressing fly under Tub5‐GS driver (Log‐rank Mantel–Cox test; n = 71–93/genotype) with control or SRSF1 siRNAs. ** P < 0.01. Data information: For eye scoring, target siRNA lines were compared to non‐targeting control siRNA lines using a two‐tailed Student’s t ‐test with Welch’s correction for multiple comparisons. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Human orthologs of fly genes are used for labeling. Details of fly genes are described in Appendix Table . Source data are available online for this figure.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Construct, Quantitation Assay, Expressing, Over Expression, Luciferase, Two Tailed Test, Labeling

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A–C Survival assays of (CGG)90‐EGFP and (G4C2)28‐EGFP expressing fly under Tub5‐GS driver with respective siRNAs as mentioned. Log‐rank Mantel–Cox test; n = 50–61 (A), 65–67 (B); and 65–73 (C). * P < 0.05. D Drosophila SRSF1 (dSF2, blue bar) and SRPK1 (gray) levels after siRNA knockdown as compared to non‐targeting siRNA (siNTC, black bars), quantified by qRT–PCR. Error bars represent mean ± SD RNAs from two ( n = 2 biological repeats) independent fly crosses (20–25 flies/genotype per cross) used to run RT–qPCR in triplicates (3 technical replicates). All data points presented in the graphs. E Western blot of FMR‐polyG‐EGFP RAN products in (CGG)90‐EGFP/ELAV‐GS flies with or without SRSF1 knockdown. Error bars represent mean ± SD. Total protein from three ( n = 3 biological repeats) independent fly crosses (20–22 flies/genotype per cross) and run in four replicates per gel (technical replicates). All data points presented in the graphs. Data information: Statistical analysis in (D) and (E) is performed using Student’s t ‐test with Welch’s correction. * P < 0.05, **** P < 0.0001.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A, B Representative (A) photographs of fly eyes and (B) quantitation with siRNA‐mediated knockdown of SRPK1 (dSRPK1); n = 30–34/genotype. Error bars represent mean ± SD. C Representative photographs of fly eyes expressing GMR‐GAL4‐driven (GGGGCC)28‐EGFP with siRNA‐mediated knockdown of SRPK1 or disruption by insertion. D Quantitation of rough eye phenotypes. t ‐test with Welch corrections for comparisons with the control; n = 31–34 flies/ genotype. Error bars represent mean ± SD. E Representative external eye imaging for the detection of GFP aggregates caused by (CGG)90‐EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). F Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90‐EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n = 13–15 flies/genotype. Error bars represent mean ± SD. Data information: Statistical analysis was performed using two‐tailed Student’s t ‐test with Welch’s correction, *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Quantitation Assay, Expressing, Imaging, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A Schematic of SRPK1 signaling pathway that regulates subcellular SRSF1 localization, which in turns can impact export and translation of repeat RNAs in the cytoplasm. SRPK1 may also act directly on protein translation pathways (dotted arrow). Known pharmacological compounds that inhibit SRPK1 can disrupt this pathway. B Anti‐FLAG immunoblot of DMSO and SRPIN340 pre‐treated HEK293T cells expressing AUG‐nLuc‐3xFLAG control or CGG‐nLuc‐3xFLAG RAN translation reporters. β‐Actin is used as a loading control. To prevent signal saturation, AUG‐nLuc lysate was diluted 1:3 in sample buffer prior to loading ( n = 3 biological replicates). Schematics of the AUG‐nLUC‐3xFLAG and +1CGG(100)‐nLuc‐3xFLAG reporters presented on top. C Relative expression of AUG‐nLuc and CGG‐nLuc reporters in HEK293T cells ( n = 8–9 biological replicates) following treatment with DMSO and SRPIN340. D Anti‐FLAG immunoblot of DMSO and SRPIN340 pre‐treated HEK293T cells expressing GGGGCC‐nLuc‐3xFLAG (GA70) and +2CGG‐nLuc‐3xFLAG (FMRpolyA) RAN translation reporters ( n = 3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. E Immunoblot of DMSO and SPHINX31 pre‐treated HEK293T cells expressing AUG‐nLuc‐3xFLAG control or CGG‐nLuc‐3xFLAG RAN translation reporters ( n = 3 biological replicates). F Anti‐FLAG of DMSO and SPHINX31 pre‐treated HEK293T cells expressing GGGGCC‐nLuc‐3xFLAG (GA70) and +2CGG‐nLuc‐3xFLAG (FMRpolyA) RAN translation reporters ( n = 3 biological replicates). Data information: Error bars represent mean ± SD. Statistical analysis was performed using two‐tailed Student’s t ‐test with Welch’s correction. * P < 0.05; ** P < 0.01, *** P < 0.001, and **** P < 0.0001. To prevent over‐exposure, the AUG‐nLuc lysate was diluted 1:3 in sample buffer. Source data are available online for this figure.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A Immunoblot of cytoplasmic phospho‐SRSF1/2 after SRPIN340 treatment. GAPDH is used as the loading control. Error bars represent mean ± SD ( n = 3 biological replicates). Statistical analysis was performed using Student’s t ‐test with Welch’s correction. * P < 0.05. Full images of the same blot showing levels of all phospho SR proteins (pan SRSF phosphorylation) after SRPIN340 treatment are presented in Fig . B ICC images of FLAG‐tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal (see methods for details). Error bars indicate mean ± 95% CI ( n = 85–126 cells/condition). Statistical analysis was performed using t ‐test with Welch’s correction, **** P < 0.0001. C Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal (see methods for details) as parts of whole. Error bars indicate mean ± 95% CI ( n = 124–151 cells/condition). Statistical analysis was performed using t ‐test with Welch’s correction, **** P < 0.0001. D Anti‐FLAG immunoblot blot of DMSO and SRPIN340 pre‐treated HEK293T cells transfected with in vitro transcribed CGG‐nLuc‐3xFLAG reporter RNA. β‐Actin is used as a loading control. Error bars represent mean ± SD ( n = 6 biological replicates). Statistical analysis was performed using Student’s t ‐test with Welch’s correction. * P < 0.05. E Relative expression of in vitro transcribed CGG‐nLuc reporter in HEK293T cells pre‐treated with DMSO and SRPIN340. Error bars represent mean ± SD ( n = 9 biological replicates). F Expression of in vitro transcribed Aug‐nLuc and CGG‐nLuc reporters in rabbit reticulocyte lysate (RRL) in vitro translation system following pre‐treatment with DMSO or SRPIN340. Error bars represent mean ± SD ( n = 9 biological replicates). Data information: For E and F, statistical analysis was performed using Student’s t ‐test with Welch’s correction. * P < 0.05; ** P < 0.01. Source data are available online for this figure.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Western Blot, Transfection, In Vitro, Expressing

Journal: EMBO Molecular Medicine

Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity

doi: 10.15252/emmm.202114163

Figure Lengend Snippet: A Subcellular levels of phosphorylated SRSF proteins with or without SRPIN340 treatment measured by Western blot using an antibody, which detects phosphorylated SR proteins (p‐SRSFs). GAPDH is used as cytoplasmic marker and loading control for cytoplasmic fraction of phosphorylated SRSFs ( n = 3 biological repeats). Detected p‐SRSFs are labeled/color‐coded. p‐SRSF1/2 data presented in Fig are outlined in red dots. B Relative nanoluciferase (nLuc) expression of +1 CGG‐nLuc‐3xF reporter transfected as plasmid or as in vitro transcribed RNA in presence or absence of SRPIN340 treatment ( n = 9). Error bars represent mean ± SD ( n = 12 biological repeats). Statistical analysis is performed using two‐tailed Student’s t ‐test with Welch’s correction. ** P < 0.01, *** P < 0.001.

Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk.

Techniques: Western Blot, Marker, Labeling, Expressing, Transfection, Plasmid Preparation, In Vitro, Two Tailed Test