aff4  (Proteintech)

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Endogenous MeCP2 interacts with endogenous SEC subunits (AFF4, AF9, ENL, and ELL2) and RNA pol II in HEK293T cells. Normal mouse immunoglobulin G was used as a negative control. ( B ) Endogenous MeCP2 interacts with SEC subunits (AFF4 and ELL2) and RNA pol II in the cortex of WT mouse at 7 weeks of age. ( C ) Reverse IP of endogenous AFF4 from WT cortical lysate and immunoblotting against MeCP2. Different brightness settings were used for the top and bottom blots because of the relatively weaker MeCP2 co-IP band intensity compared to the AFF4 IP band intensity. Immunoblotting against AFF4 for (A) and (B) was performed with the Bethyl Laboratories antibody (A302-538A), whereas IP and immunoblotting for AFF4 for (C) was performed with the Proteintech antibody (14662-1-AP).

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Negative Control, Western Blot, Co-Immunoprecipitation Assay

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Short hairpin–mediated knockdown of AFF4 in HEK293T cells abolishes the interaction between MeCP2 and the remaining subunits of the SEC (ENL and ELL2). While knockdown of ENL mildly dissociates MeCP2’s interaction with ELL2, it does not affect its interaction with AFF4. Knockdown of ELL2 does not affect the interaction between MeCP2 and the remaining subunits of the SEC. NT sh is a nontargeting short-hairpin negative control. ( B ) In vitro binding assay between recombinant FLAG-tagged MeCP2 and HA-tagged SEC subunits. ( C ) In vitro binding assay between FLAG-tagged MeCP2 fragments and HA-tagged AFF4. The fragments that show a decrease in interaction with HA-AFF4 are highlighted in bold. Diagram on the right shows a map of the MeCP2 fragments tested. NTD, N-terminal domain; MBD, methyl-CpG–binding domain; ID, intervening domain; TRD, transcriptional repression domain; CTD, C-terminal domain. ( D ) In vitro binding assay between AFF4 and MeCP2 with deletions in the NTD, ID, TRD, ID and TRD, and CTD. The domains critical for interaction are highlighted in bold.

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Knockdown, Negative Control, In Vitro, Binding Assay, Recombinant

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Global heatmap of log 2 -transformed occupancy of AFF4 in the cortex of WT and Mecp2 null mice. ( B ) Global heatmap of log 2 fold change of AFF4 occupancy in Mecp2 null mouse compared to WT mouse. ( C ) Global heatmap of log 2 -transformed occupancy of RNA pol II in the cortex of WT and Mecp2 null mice. ( D ) Global heatmap of log 2 fold change of RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. ( E ) Global heatmap of log 2 -transformed occupancy of pSer 2 RNA pol II in the cortex of WT and Mecp2 null mice. ( F ) Global heatmap of log 2 fold change of pSer 2 RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. n = 8696 RNA pol II–bound genes are represented in all heatmaps.

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Transformation Assay

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Global heatmap showing the log 2 fold change of AFF4, RNA pol II, and pSer 2 RNA pol II binding in the Mecp2 null cortex based on hierarchical clustering. ( B ) Two-dimensional plots for clusters in (A), showing the correlation between AFF4 and RNA pol II binding as the median of Mecp2 null versus WT ratios across matched animal pairs. n = 3 biological replicates. Color scale indicates the gene count. Spearman’s correlation values: ρ = 0.081; P = 0.00019 (cluster I), ρ = 0.13; P < 2.2 × 10 −16 (cluster II), ρ = 0.34; P = 4.8 × 10 −10 (cluster III). ( C ) Two-dimensional plots for clusters in (A), showing the correlation between AFF4 binding and RNA expression as the median of Mecp2 null versus WT ratios across matched animal pairs. Color scale indicates the gene count. n = 3 biological replicates for AFF4 ChIP-seq and n = 6 biological replicates for RNA-seq. Spearman’s correlation values: cluster I ρ = 0.026, P = 0.23; cluster II ρ = 0.063, P = 7.9 × 10 −07 ; cluster III ρ = 0.27, P = 5.3e-07.

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Binding Assay, RNA Expression, ChIP-sequencing, RNA Sequencing

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Open-field assessment of activity levels and duration at the center of the arena beginning at 10 weeks of age. ( B ) Rotarod assay measures motor-coordination ability. ( C ) Fear conditioning assay measuring learning and memory capabilities in both contextual and cued settings. For open-field and fear conditioning assays [(A) and (C)], n = 32 WT mice (gray); n = 16 Aff4 heterozygous knockout mice (blue); n = 36 Mecp2 hypomorphic mice (orange); n = 22 double-mutant mice (green). For rotarod (B), n = 22 WT mice; n = 14 Aff4 heterozygous knockout mice; n = 29 Mecp2 hypomorphic mice; n = 19 double-mutant mice. ns, not significant; * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Activity Assay, Knock-Out, Mutagenesis

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Endogenous MeCP2 interacts with endogenous SEC subunits (AFF4, AF9, ENL, and ELL2) and RNA pol II in HEK293T cells. Normal mouse immunoglobulin G was used as a negative control. ( B ) Endogenous MeCP2 interacts with SEC subunits (AFF4 and ELL2) and RNA pol II in the cortex of WT mouse at 7 weeks of age. ( C ) Reverse IP of endogenous AFF4 from WT cortical lysate and immunoblotting against MeCP2. Different brightness settings were used for the top and bottom blots because of the relatively weaker MeCP2 co-IP band intensity compared to the AFF4 IP band intensity. Immunoblotting against AFF4 for (A) and (B) was performed with the Bethyl Laboratories antibody (A302-538A), whereas IP and immunoblotting for AFF4 for (C) was performed with the Proteintech antibody (14662-1-AP).

Article Snippet: Membranes were blocked for 30 min using the INTERCEPT (TBS) blocking buffer (Li-Cor, 927-60010) and then incubated with the following primary antibodies overnight at 4°C: AFF4 (Bethyl Laboratories, A302-538A at 1:1000; Proteintech, 14662-1-AP at 1:5000; and Boster Bio, A03824 at 1:1000), AF9 (Genetex, GTX 102835 at 1:1000), ENL (Cell Signaling Technology; 14893 at 1:1000), ELL2 (Bethyl; A302-505A at 1:1000), total RNA pol II (Cell Signaling Technology; 14958S at 1:1000), pSer 2 RNA pol II (Millipore, 04-1571 at 1:1000), MeCP2 (Cell Signaling Technology; 3456S at 1:1000), GFP (Abcam; ab13970 at 1:10,000), β-actin (Cell Signaling Technology; 8457S at 1:10,000), vinculin (Sigma-Aldrich; V9131-.2ML at 1:20,000), FLAG (Sigma-Aldrich; F3165-1MG at 1:4000), and HA (Cell Signaling Technology; 3724S at 1:4000).

Techniques: Negative Control, Western Blot, Co-Immunoprecipitation Assay

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Short hairpin–mediated knockdown of AFF4 in HEK293T cells abolishes the interaction between MeCP2 and the remaining subunits of the SEC (ENL and ELL2). While knockdown of ENL mildly dissociates MeCP2’s interaction with ELL2, it does not affect its interaction with AFF4. Knockdown of ELL2 does not affect the interaction between MeCP2 and the remaining subunits of the SEC. NT sh is a nontargeting short-hairpin negative control. ( B ) In vitro binding assay between recombinant FLAG-tagged MeCP2 and HA-tagged SEC subunits. ( C ) In vitro binding assay between FLAG-tagged MeCP2 fragments and HA-tagged AFF4. The fragments that show a decrease in interaction with HA-AFF4 are highlighted in bold. Diagram on the right shows a map of the MeCP2 fragments tested. NTD, N-terminal domain; MBD, methyl-CpG–binding domain; ID, intervening domain; TRD, transcriptional repression domain; CTD, C-terminal domain. ( D ) In vitro binding assay between AFF4 and MeCP2 with deletions in the NTD, ID, TRD, ID and TRD, and CTD. The domains critical for interaction are highlighted in bold.

Article Snippet: Membranes were blocked for 30 min using the INTERCEPT (TBS) blocking buffer (Li-Cor, 927-60010) and then incubated with the following primary antibodies overnight at 4°C: AFF4 (Bethyl Laboratories, A302-538A at 1:1000; Proteintech, 14662-1-AP at 1:5000; and Boster Bio, A03824 at 1:1000), AF9 (Genetex, GTX 102835 at 1:1000), ENL (Cell Signaling Technology; 14893 at 1:1000), ELL2 (Bethyl; A302-505A at 1:1000), total RNA pol II (Cell Signaling Technology; 14958S at 1:1000), pSer 2 RNA pol II (Millipore, 04-1571 at 1:1000), MeCP2 (Cell Signaling Technology; 3456S at 1:1000), GFP (Abcam; ab13970 at 1:10,000), β-actin (Cell Signaling Technology; 8457S at 1:10,000), vinculin (Sigma-Aldrich; V9131-.2ML at 1:20,000), FLAG (Sigma-Aldrich; F3165-1MG at 1:4000), and HA (Cell Signaling Technology; 3724S at 1:4000).

Techniques: Knockdown, Negative Control, In Vitro, Binding Assay, Recombinant

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Global heatmap of log 2 -transformed occupancy of AFF4 in the cortex of WT and Mecp2 null mice. ( B ) Global heatmap of log 2 fold change of AFF4 occupancy in Mecp2 null mouse compared to WT mouse. ( C ) Global heatmap of log 2 -transformed occupancy of RNA pol II in the cortex of WT and Mecp2 null mice. ( D ) Global heatmap of log 2 fold change of RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. ( E ) Global heatmap of log 2 -transformed occupancy of pSer 2 RNA pol II in the cortex of WT and Mecp2 null mice. ( F ) Global heatmap of log 2 fold change of pSer 2 RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. n = 8696 RNA pol II–bound genes are represented in all heatmaps.

Article Snippet: Membranes were blocked for 30 min using the INTERCEPT (TBS) blocking buffer (Li-Cor, 927-60010) and then incubated with the following primary antibodies overnight at 4°C: AFF4 (Bethyl Laboratories, A302-538A at 1:1000; Proteintech, 14662-1-AP at 1:5000; and Boster Bio, A03824 at 1:1000), AF9 (Genetex, GTX 102835 at 1:1000), ENL (Cell Signaling Technology; 14893 at 1:1000), ELL2 (Bethyl; A302-505A at 1:1000), total RNA pol II (Cell Signaling Technology; 14958S at 1:1000), pSer 2 RNA pol II (Millipore, 04-1571 at 1:1000), MeCP2 (Cell Signaling Technology; 3456S at 1:1000), GFP (Abcam; ab13970 at 1:10,000), β-actin (Cell Signaling Technology; 8457S at 1:10,000), vinculin (Sigma-Aldrich; V9131-.2ML at 1:20,000), FLAG (Sigma-Aldrich; F3165-1MG at 1:4000), and HA (Cell Signaling Technology; 3724S at 1:4000).

Techniques: Transformation Assay

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Global heatmap showing the log 2 fold change of AFF4, RNA pol II, and pSer 2 RNA pol II binding in the Mecp2 null cortex based on hierarchical clustering. ( B ) Two-dimensional plots for clusters in (A), showing the correlation between AFF4 and RNA pol II binding as the median of Mecp2 null versus WT ratios across matched animal pairs. n = 3 biological replicates. Color scale indicates the gene count. Spearman’s correlation values: ρ = 0.081; P = 0.00019 (cluster I), ρ = 0.13; P < 2.2 × 10 −16 (cluster II), ρ = 0.34; P = 4.8 × 10 −10 (cluster III). ( C ) Two-dimensional plots for clusters in (A), showing the correlation between AFF4 binding and RNA expression as the median of Mecp2 null versus WT ratios across matched animal pairs. Color scale indicates the gene count. n = 3 biological replicates for AFF4 ChIP-seq and n = 6 biological replicates for RNA-seq. Spearman’s correlation values: cluster I ρ = 0.026, P = 0.23; cluster II ρ = 0.063, P = 7.9 × 10 −07 ; cluster III ρ = 0.27, P = 5.3e-07.

Article Snippet: Membranes were blocked for 30 min using the INTERCEPT (TBS) blocking buffer (Li-Cor, 927-60010) and then incubated with the following primary antibodies overnight at 4°C: AFF4 (Bethyl Laboratories, A302-538A at 1:1000; Proteintech, 14662-1-AP at 1:5000; and Boster Bio, A03824 at 1:1000), AF9 (Genetex, GTX 102835 at 1:1000), ENL (Cell Signaling Technology; 14893 at 1:1000), ELL2 (Bethyl; A302-505A at 1:1000), total RNA pol II (Cell Signaling Technology; 14958S at 1:1000), pSer 2 RNA pol II (Millipore, 04-1571 at 1:1000), MeCP2 (Cell Signaling Technology; 3456S at 1:1000), GFP (Abcam; ab13970 at 1:10,000), β-actin (Cell Signaling Technology; 8457S at 1:10,000), vinculin (Sigma-Aldrich; V9131-.2ML at 1:20,000), FLAG (Sigma-Aldrich; F3165-1MG at 1:4000), and HA (Cell Signaling Technology; 3724S at 1:4000).

Techniques: Binding Assay, RNA Expression, ChIP-sequencing, RNA Sequencing

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Open-field assessment of activity levels and duration at the center of the arena beginning at 10 weeks of age. ( B ) Rotarod assay measures motor-coordination ability. ( C ) Fear conditioning assay measuring learning and memory capabilities in both contextual and cued settings. For open-field and fear conditioning assays [(A) and (C)], n = 32 WT mice (gray); n = 16 Aff4 heterozygous knockout mice (blue); n = 36 Mecp2 hypomorphic mice (orange); n = 22 double-mutant mice (green). For rotarod (B), n = 22 WT mice; n = 14 Aff4 heterozygous knockout mice; n = 29 Mecp2 hypomorphic mice; n = 19 double-mutant mice. ns, not significant; * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Membranes were blocked for 30 min using the INTERCEPT (TBS) blocking buffer (Li-Cor, 927-60010) and then incubated with the following primary antibodies overnight at 4°C: AFF4 (Bethyl Laboratories, A302-538A at 1:1000; Proteintech, 14662-1-AP at 1:5000; and Boster Bio, A03824 at 1:1000), AF9 (Genetex, GTX 102835 at 1:1000), ENL (Cell Signaling Technology; 14893 at 1:1000), ELL2 (Bethyl; A302-505A at 1:1000), total RNA pol II (Cell Signaling Technology; 14958S at 1:1000), pSer 2 RNA pol II (Millipore, 04-1571 at 1:1000), MeCP2 (Cell Signaling Technology; 3456S at 1:1000), GFP (Abcam; ab13970 at 1:10,000), β-actin (Cell Signaling Technology; 8457S at 1:10,000), vinculin (Sigma-Aldrich; V9131-.2ML at 1:20,000), FLAG (Sigma-Aldrich; F3165-1MG at 1:4000), and HA (Cell Signaling Technology; 3724S at 1:4000).

Techniques: Activity Assay, Knock-Out, Mutagenesis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: N6-methyladenosine-modified circCDK14 promotes ossification of the ligamentum flavum via epigenetic modulation by targeting AFF4

doi: 10.1007/s00018-024-05460-4

Figure Lengend Snippet: MiR-93-5p repressed the AFF4 expression by targeting the 3’-UTR of AFF4. A Bioinformatic analysis indicating that AFF4 is a potential target of miR-93-5p. B , C mRNA ( B ) and protein ( C ) levels of AFF4 in LF or OLF tissues (Scale bar = 50 μm). D Luciferase reporter activity between AFF4 and miR-93-5p. E , G mRNA ( E ) and protein levels ( F , G ) of AFF4 after the transfection of miR-93-5p mimics. H mRNA level of AFF4 after the transfection of siRNAs targeting AFF4. I – K Protein level of osteogenic markers (OPN and RUNX2) after the AFF4 knockdown. L – O ALP ( L , M ) and ARS ( N , O ) intensity after the AFF4 knockdown (Scale bar = 25 μm). LF , ligamentum flavum; OLF , ossification of the ligamentum flavum; ALP , alkaline phosphatase; ARS , Alizarin Red S; * p < 0.05, ** p < 0.01, *** p < 0.001, ns , not significant

Article Snippet: The details of the preliminary antibodies used in this study were as follows: OPN (ImmunoWay, USA), RUNX2 (Abcam, USA), AFF4 (Proteintech, USA), AGO2 (Cell Signaling Technology, USA), and WTAP (Abcam, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Knockdown

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: N6-methyladenosine-modified circCDK14 promotes ossification of the ligamentum flavum via epigenetic modulation by targeting AFF4

doi: 10.1007/s00018-024-05460-4

Figure Lengend Snippet: CircCDK14 modulated the osteogenesis of LF cells via the miR-93-5p/AFF4 pathway ( A – C ) mRNA ( A ) and protein levels ( B , C ) of AFF4 after the co-transfection of circCDK14 overexpression vector and miR-93-5p mimics. ( D – F ) Protein levels of osteogenic markers (OPN and RUNX2) after the co-transfection of circCDK14 overexpression vector and miR-93-5p mimics ( G – J ) ALP ( G , H ) and ARS ( I , J ) intensity after the co-transfection of circCDK14 overexpression vector and miR-93-5p mimics (Scale bar = 25 μm). OE , overexpression; ALP , alkaline phosphatase; ARS , Alizarin Red S; * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001; ns , not significant

Article Snippet: The details of the preliminary antibodies used in this study were as follows: OPN (ImmunoWay, USA), RUNX2 (Abcam, USA), AFF4 (Proteintech, USA), AGO2 (Cell Signaling Technology, USA), and WTAP (Abcam, USA).

Techniques: Cotransfection, Over Expression, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: N6-methyladenosine-modified circCDK14 promotes ossification of the ligamentum flavum via epigenetic modulation by targeting AFF4

doi: 10.1007/s00018-024-05460-4

Figure Lengend Snippet: Schematic representation of the investigated mechanisms. The WTAP expression was significantly increased in OLF tissues. The increased WTAP expression elevated the m6A modification level in circCDK14, which was further recognized by the IGF2BP3 and promoted the stability of circCDK14. The increased circCDK14 expression further stimulated the osteogenic differentiation of LF cells via the miR-93-5p/AFF4 pathway

Article Snippet: The details of the preliminary antibodies used in this study were as follows: OPN (ImmunoWay, USA), RUNX2 (Abcam, USA), AFF4 (Proteintech, USA), AGO2 (Cell Signaling Technology, USA), and WTAP (Abcam, USA).

Techniques: Expressing, Modification

Journal: bioRxiv

Article Title: Inhibition of SF3B1 affects recruitment of P-TEFb to chromatin through multiple mechanisms

doi: 10.1101/2024.06.26.600844

Figure Lengend Snippet: ( A )Western blots of SNW1, U1-A (loading control), histone H3 (loading control), and β-tubulin (loading control) from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells treated with DMSO, ethanol, 0.02 µM Leptomycin B, or 1 µM PlaB. Treatments were performed as follow: Ethanol 1h/DMSO 30 min (-/-), LMB 1h/DMSO 30 min (+/-), Ethanol 1h/PlaB 30 min (-/+), LMB 1h/PlaB 30 min (+/+). ( B ) Total RNAPII, BRD4, AFF4, MYC, and HTATSF1 ChIP-qPCR on the promoter regions of KPNB1, CCND2, LDLR , and SLCO4A1 in HeLa cells treated with DMSO or 1 µM PlaB for 30 min. Statistical test: Unpaired t-test, n = 3-4 biological replicates. P-value: * < 0.05, *** < 0.001. The RNAPII ChIP-qPCR data for TSS, TSS+3.2, LDLR, and SLCO4A1 are the same as the ones shown in Supplementary Figure 7C (same ChIP-qPCR experiments). ( C ) Western blots of NRD4, AFF4, MYC, histone H3 (loading control), and Nucleolin (loading control) from chromatin and nucleoplasm fractions of HeLa cells treated with DMSO or 1 µM PlaB for 30 min. ( D ) Western blots of SRSF2 and β-actin (loading control) from whole cell extract of HeLa cells treated with DMSO or 1 µM PlaB for 30 min. ( E ) Representative images of immunofluorescence analysis of SRSF2 in HeLa cells treated with DMSO, 1 µM Bortezomib, or 1 µM PlaB. Treatments were performed as follow: DMSO 1h/DMSO 30 min (DMSO), Bortezomib 1h/DMSO 30 min (Bortezomib), DMSO 1h/PlaB 30 min (PlaB), Bortezomib 1h/PlaB 30 min (Bortezomib & PlaB). SRSF2 (green), DAPI (blue), scale bars: 50 µm. ( F ) Quantification of SRSF2 fluorescent intensity for DMSO (blue), Bortezomib (orange), PlaB (red), and Bortezomib & PlaB (purple). Boxplot settings are: min to max values with the box showing 25-75 percentile range. 10,106 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001.

Article Snippet: Western blots were performed with NuPAGE Novex 4–12% Bis–Tris Protein Gels (Life Technologies) with the following primary antibodies: Rpb1 NTD (D8L4Y) Rabbit mAb (14958S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Rabbit anti-SF3b155/SAP155 (A300-996A, Bethyl Laboratories), Rabbit anti-Phospho-SF3B1 Ser129 (PD043, MBL International), Rabbit anti-Phospho-SF3B1 Thr142 (( )), Rabbit anti-Phospho-SF3B1 Thr211 (PA5-105427, Invitrogen), Rabbit anti-Phospho-SF3B1 Thr313 (D8D8V, Cell Signaling), Rabbit anti-Tat-SF1 (A302-023A, Bethyl Laboratories), Rabbit anti-CDK9 (ab76320, Abcam), Rabbit anti-Phospho-CDK9 (pThr186) (2549, Cell Signaling), Rabbit anti-Cyclin T1 (ab184703, Abcam), Rabbit anti-SNW1 (A300-784A, Bethyl Laboratories), Rabbit anti-c-MYC (sc-764, Santa Cruz Biotechnology), Rabbit anti-SC35 (SRSF2) (ab204916, Abcam), Rabbit anti-BRD4 (A301-985A100, Bethyl Laboratories), Rabbit anti-MCEF (AFF4) (A302-539A, Bethyl Laboratories), Mouse anti-U1A (3F9-1F7, Novus Biologicals), Rabbit anti-Actin (C-11) (sc-1615, Santa Cruz Biotechnology), Rabbit anti-α-Tubulin (2144S, Cell Signaling), Rabbit anti-Nucleolin (ab305947), Abcam), and Histone H3 (ab1791, Abcam).

Techniques: Western Blot, Control, ChIP-qPCR, Immunofluorescence