anti p53  (Bio-Rad)

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Moderate regulation of alternatively spliced p53β mRNA by UPF1. A Alternative splicing (AS) and nonsense-mediated decay (NMD) involving exon 9b (E9b) within p53 intron 9. Inclusion or skipping of E9b (dark gray box) generates p53β or p53α mRNA, respectively. E9b is expected to recruit UPF1 and can be degraded. B UV-crosslinking RNA immunoprecipitation (UV-RIP) assays. HeLa cells were cotransfected with Flag-UPF1 and Dup-p53 minigenes. Lysates were immunoprecipitated using anti-Flag M2 agarose beads. Enrichment of the E9b-included isoform (Dup-β) was assessed using primers spanning the E9b–β-globin exon 2 junction of p53. Data are represented as mean ± SEM (n ≥ 3). * p < 0.05. C Splicing analysis of Dup-p53 minigene-transfected HeLa cells. STOP and GO reporters were used for NMD analysis with or without a premature termination codon (PTC). Primers targeting β-globin exon 1 (forward) and exon 2 (reverse) were used. The upper band corresponds to the E9b-included isoform (β), and the lower band to the E9b-skipped isoform (α). β-actin serves as a loading control. D Splicing analysis from Dup-p53 (STOP) minigene in siUPF1-transfected HeLa cells compared with siGFP-transfected control cells. The upper band corresponds to the E9b-included isoform (β), and the lower band to the E9b-skipped isoform (α). RT-PCR was performed employing β-globin exon 1 (forward) and exon 2 (reverse) primers. UPF1 knockdown was confirmed by Western blot assays (bottom). β-actin was used as a loading control. E RT-qPCR analysis for endogenous p53 mRNA isoforms—p53α and p53β—in UPF1-depleted HeLa cells. F RT-qPCR analysis for exon 1 of endogenous p53 in UPF1-depleted HeLa cells, corresponding to the 5′ UTR. (G, H) Decay rates of p53α mRNA G and p53β mRNA H in siUPF1- and siGFP-transfected cells following transcription inhibition using actinomycin D (ActD). The mean half-lives of p53α or p53β mRNAs are indicated by a dashed line. Data are represented as mean ± SEM (n ≥ 3)

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Alternative Splicing, RNA Immunoprecipitation, Immunoprecipitation, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Knockdown, Western Blot, Quantitative RT-PCR, Inhibition

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Identification and localization of the intron-retained p53 transcript. A Diagram of sequential (seq) or non-sequential (non-seq) splicing of p53 intron 9. I9a and I9b indicate the upstream and downstream introns flanking E9b, respectively. Predicted premature termination codons (PTCs) are shown in red. B RT-qPCR analysis of intron retention in p53 transcripts under basal conditions in HeLa cells. I2, intron2; N.D., not detected. C Decay rates of p53-IR transcript in siUPF1- and siGFP-transfected HeLa cells following transcription inhibition with ActD. The mean half-lives of p53-IR transcript are indicated by a dashed line. Data are represented as mean ± SEM (n ≥ 3). D RT-qPCR-based quantification of p53α, p53β, and p53-IR transcript following cycloheximide (CHX) treatment in HeLa cells. Data are represented as mean ± SEM (n ≥ 3). ** p < 0.01. E , F Ratios of p53α, p53β, and p53-IR transcript levels altered by CHX treatment in the nuclear and cytoplasmic fractions of HeLa cells, compared with DMSO-treated controls. Data are presented as mean ± SEM (n ≥ 3). *** p < 0.001. G Subcellular localization of p53 transcripts in HeLa cells. Cytoplasmic (Cy), Nucleoplasmic (Np), and chromatin-associated (Chr) fractions are indicated. GAPDH pre-mRNA and NEAT1 long noncoding RNA were used as nuclear-retained RNA markers, and 18S ribosomal RNA was used as a cytoplasmic RNA marker

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Quantitative RT-PCR, Transfection, Inhibition, Marker

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Chromatin association of SRSF3 and UPF1 at the TP53 gene. A Subcellular localization of SRSF3 and UPF1 in HeLa cells. α-tubulin and Histone H3 are markers for the cytoplasmic and chromatin-associated fractions, respectively. Regarding SRSF1, hyperphosphorylated forms localize to the nucleoplasm, while hyperphosphorylated forms localize to the cytoplasm. B UPF1 and SRSF3 chromatin immunoprecipitation (ChIP) analysis of TP53 genomic regions. UPF1 ChIP was performed using an anti-UPF1 antibody compared with an anti-IgG control in HeLa cells, whereas SRSF3 ChIP was performed using an anti-Flag antibody in Flag-SRSF3-expressing HeLa cells compared with Flag-empty vector (Flag-Vec) control cells. Pro, promoter; I4, intron 4; I9, intron 9; E11, exon 11. Data are presented as mean ± SEM (n ≥ 3). * p < 0.05; ** p < 0.01. C Coimmunoprecipitation (co-IP) showing the interaction between UPF1 and SRSF3. HeLa cells were cotransfected with Myc-UPF1 and Flag-SRSF3, and immunoprecipitation was performed utilizing anti-Flag antibody. D Coimmunofluorescence (co-IF) of UPF1 and SRSF3 after treatment with transcription or translation inhibitors, which are actinomycin D (ActD) and flavopiridol (Flavo), or cycloheximide (CHX), respectively. UPF1 was detected using Cy3-conjugated secondary antibody (red), and SRSF3 was identified employing the Alexa Fluor 488-conjugated one (green). Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm. E Quantification of UPF1 and SRSF3 colocalization by Pearson’s correlation coefficient. Data are presented as mean ± SEM (n ≥ 30). *** p < 0.001. F Representative images of in situ proximity ligation assay (PLA) between UPF1 and SRSF3 in HeLa cells. Each cell was treated with DMSO (control), ActD, Flavo, or CHX. PLA signals are shown as red puncta, and nuclei are counterstained with DAPI (blue). Scale bars, 5 μm. G Quantification of the mean of nuclear PLA signals per nucleus. Data are presented as mean ± SEM (n ≥ 30). Statistical significance was determined by one-way ANOVA. * p < 0.05; *** p < 0.001

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Chromatin Immunoprecipitation, Control, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation, In Situ, Proximity Ligation Assay

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Binding of SRSF3 at the retained intron of p53 transcript. A UV-RNA immunoprecipitation (UV-RIP) analysis of endogenous p53 (pre-)mRNA. Flag-SRSF3-expressing and Flag-Vec control cells were subjected to immunoprecipitation using an anti-Flag antibody. Data are presented as mean ± SEM (n ≥ 3). * p < 0.05; ** p < 0.01. B Schematic diagram of Dup-p53 minigene reporters harboring mutations in SRSF3-binding motifs, indicated by black circles, and mutated motifs by white circles. The WT contains all intact SRSF3-binding motifs. Mutant reporters (Mut1–Mut4) contain individual or combinations of mutated SRSF3-binding motifs, as indicated. C UV-RIP analysis of Dup-p53 minigene reporters. Flag-SRSF3-expressing cells and Flag-Vec control cells were subjected to immunoprecipitation using an anti-Flag antibody. Data are presented as mean ± SEM (n ≥ 3). ** p < 0.01. D Splicing analysis of Dup-p53 minigene reporters in transfected HeLa cells. Relative Dup-β transcript level was measured by RT-qPCR. Data are presented as mean ± SEM (n ≥ 3). * p < 0.05. E SRSF3 tethering analysis in combination with UPF1 knockdown. MS2 RNA hairpins were inserted into I9a of the Dup-p53 minigene, and MS2-binding protein (MBP)-fused SRSF3 was co-transfected to tether SRSF3 to the MS2 RNA. RT-qPCR was used to quantify Dup-α and Dup-β isoforms. Data are presented as mean ± SEM (n ≥ 3). ** p < 0.01. F Proposed model illustrating the splicing-linked RNA surveillance mechanism. p53-IR transcript interacts with SRSF3, which subsequently recruits UPF1. Formation of p53-IR–SRSF3–UPF1 ternary complex suppresses E9b inclusion, thereby preventing production of the p53β mRNA

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Binding Assay, RNA Immunoprecipitation, Expressing, Control, Immunoprecipitation, Mutagenesis, Transfection, Quantitative RT-PCR, Knockdown

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Expression of C-terminal truncated p53 protein upon SRSF3 depletion. A RT-qPCR analysis of p53α and p53β mRNA levels in HEK293, HeLa, HCT116, and SW480 cells transfected with siRNA targeted to SRSF3 (siSRSF3) compared to control siRNA (siGFP). Exon junction-specific primers used for isoform detection are shown (left). Data are presented as mean ± SEM (n ≥ 3). * p < 0.05; ** p < 0.01. B Western blot analysis of p53 protein isoforms in SW480 cells transfected with siRNAs targeting SRSF3 and SRSF1. p53 isoforms were detected using anti-p53 (DO-1) antibody. The upper band corresponds to p53α protein (~ 53 kDa), and the lower band to p53β protein isoform (~ 47 kDa). β-actin serves as a loading control. C Western blot analysis of p53 protein isoforms in SRSF3 knockout (KO) SW480 cells obtained by employing CRISPR/Cas9. Total cell lysates were analyzed using anti-p53 (Pab1801 and DO-11) and a custom anti-p53β-specific antibody. β-actin serves as a loading control. D Western blot analysis of p53 protein isoforms in UPF1-depleted SW480 cells (siUPF1) compared to control cells (siGFP). The anti-p53 (DO-1) antibody used in (B) was also employed to detect p53α and p53β protein isoforms. β-actin serves as a loading control. E Quantification of p53α and p53β protein isoform levels detected by anti-p53 (DO-11) antibody. Data are presented as mean ± SEM (n = 3). ** p < 0.01. F Proposed model explaining the production of the C-terminal truncated p53β protein isoform

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Knock-Out, CRISPR

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Oncogenic functions of the C-terminal truncated p53β protein isoform. A Cell morphology of colorectal cancer cell lines (HCT116 WT and HCT116 p53−/− , DLD-1) and a lung cancer cell line (A549) upon Flag-p53β overexpression. B RT-qPCR analysis of EMT markers, ZEB1 and TWIST mRNAs, in stably Flag-p53β- expressing HCT116 WT and HCT116 p53−/− , and A549 cells. Data are presented as mean ± SEM (n ≥ 3). * p < 0.05; ** p < 0.01. C , D Wound closure assay in stably Flag-p53β-expressing HCT116 WT and HCT116 p53−/− , and A549 cells. Wound closure was measured 48 h after scratching. Data are presented as mean ± SEM (n ≥ 3). * p < 0.05. E , F Matrigel invasion assay using stably Flag-p53β-expressing HCT116 WT and HCT116. p53−/− , and A549 cells. Cells were seeded in the upper chamber of Matrigel-coated transwells. Invaded cells on the bottom surface were counted after 48 h. Data are presented as mean ± SEM (n ≥ 3). * p < 0.05; *** p < 0.001

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Over Expression, Quantitative RT-PCR, Stable Transfection, Expressing, Wound Closure Assay, Invasion Assay

Journal: Cell & Bioscience

Article Title: Regulation of oncogenic C-terminal truncated p53β protein isoform expression by SRSF3–UPF1 splicing and surveillance axis

doi: 10.1186/s13578-026-01556-5

Figure Lengend Snippet: Biogenesis of p53 mRNAs and functions of p53 protein isoforms. The scheme represents the role of a newly identified intron-retained p53 transcript (p53-IR transcript) in preventing the generation of the C-terminal truncated and pro-metastatic p53β protein. During transcription elongation, SRSF3 associates with the p53-IR transcript and recruits UPF1 forming a transcript surveillance complex. It specifically monitors p53 pre-mRNA and suppress p53β mRNA expression and C-terminal-truncated p53β protein, which promote epithelial–mesenchymal transition (EMT) and metastasis

Article Snippet: The antibodies used were as follows- anti-UPF1 (Cell Signaling, #12,040), anti-SRSF3 (Invitrogen, #33–4200), anti-FLAG-M2 (Sigma, #F3165), anti-SRSF1 (Thermo Fisher, #32–4500), anti-Histone H3 (Abcam, #ab1791), anti-α-tubulin (Santa Cruz, #sc-8035), anti-β-actin (Abcam, #ab6276), anti-Myc (Sigma, #05-724MG), anti-p53 (Santa Cruz, DO-1 #sc-126; Pab1801 #sc-98; BIORAD, DO-11 #MCA1704), and E-cadherin (Biosciences, #610,181).

Techniques: Expressing

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: Western blot analyses of lamin A/C and progerin, full-length p53, Δ133p53α, and p21 Waf1/Cip1 were performed in four 15-week-old Δ133p53α-expressing Group-1 mice ( CAG-133 Tam/+ ; Cre Tg/+ ; Lmna G609G/+ ), along with four each of age-matched, non-expressing control Group-2 ( CAG-133 LSL/+ ; Cre Tg/+ ; Lmna G609G/+ ) and Group-4 mice ( CAG-133 +/+ ; Cre +/+ ; Lmna G609G/+ ), as well as two age-matched wild-type mice ( CAG-133 +/+ ; Cre +/+ ; Lmna +/+ ). Results from skin ( a ), skeletal muscle ( b ), kidney ( c ), spleen ( d ), and lung ( e ) are presented. An inter-blot control (liver from a Group-1 mouse) was included in all blots. F, female; M, male. GAPDH was a loading control and used for normalization of p21 Waf1/Cip1 , progerin, and full-length p53 expression levels. Quantitative data summaries of p21 Waf1/Cip1 , progerin, and full-length p53 in Group-1, -2 and -4 mice are shown as relative values to Group-2 mice (mean ± s.d. from n = 4; open circles indicate two females, and closed circles indicate two males). P values were determined by Welch’s t -test. Two wild-type mice were used only as references and not for statistical comparisons.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: a,b , Top Hallmark pathways identified by Gene set enrichment analysis (GSEA). The bulk RNA-seq data were obtained from the heart ( a ) and kidney ( b ) in 9-10-month-old Group-1 and Group-3 mice (n = 5 each). Pathways are ranked by normalized enrichment score. False discovery rate < 0.10. c-g, Enrichment plots illustrate significant downregulation of the p53 pathway ( c,d ) and upregulation of the oxidative phosphorylation ( e,f ) in both heart and kidney, as well as upregulation of the glycolysis in the kidney ( g ). Enrichment plots depict running enrichment scores (ES) and the distribution of genes within each Hallmark gene set. All leading edge genes in each pathway are listed in Extended Data Table 2. h,i, qRT-PCR assays of mRNA expression of genes in the oxidative phosphorylation pathway (Ndufs6, Ndufc2, Uqcrq, Hsd17b10, and Gpx4) and an antioxidant gene Prdx1 in the heart ( h ) and kidney ( i ) of 9-10-month-old Group-1 and Group-3 mice (mean ± s.d. from n = 5, each with technical triplicate; open circles, females; closed circles, males). P values were calculated by Welch’s t -test.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: RNA Sequencing, Phospho-proteomics, Quantitative RT-PCR, Expressing