importin β1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Two sequential waves of mRNA translation drive embryonic development

doi: 10.1101/2025.09.18.676998

Figure Lengend Snippet: a Double staining of GFP-Ewsr1b (green) and GFP-Ewsr1b mRNA (red) in embryos injected with GFP-Ewsr1b mRNA carrying Long-3′UTR (Long) or Short-3′UTR (Short) at 4 hpf. Scale bars: 20 µm. b Violin plots showing distances from the nuclear center to signals of GFP-Ewsr1b mRNA carrying Long-3′UTR or Short-3′UTR (means ± SD; n = 80). Similar results were obtained from two independent experiments. **********p < 0.0000000001 (Student’s t -test). c Immunofluorescence of Importin β1 in embryos at 3 hpf. d Immunoblotting of embryos at 3 hpf following IP with control IgG (IgG) or anti-Importin β1 (α-Im β) antibody, and RT-PCR for ewsr1b -3′Long and α-tubulin mRNAs. Similar results were obtained from two independent experiments. e Double staining of Importin β1 (red) and the ewsr1b -3′Long mRNA 3′UTR (green) in embryos at 3 hpf. Left: High-resolution confocal image; right: enlarged views of the boxed area. Similar results were obtained from two independent experiments. f Immunofluorescence of Importin β1 and Ewsr1b in uninjected embryos (Control) or embryos injected with Importazole at 3 hpf. DNA is shown in blue. Enlarged views of the boxed area with or without DNA staining are shown on the right side. Scale bars, 10 µm. g Quantification of average signal intensity in the nucleus per 25 µm 2 . (means ± SD; n = 10). ***********p < 0.00000000001 (Student’s t -test).

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto Immobilon membranes, and probed with primary antibodies; mouse anti-Syncrip antibody (1:1,000, hnRNP Q; Santa Cruz Biotechnology, I8E4; sc-56703), rabbit anti-Syncrip antibody (1:1,000, Proteintech, 14024-1-AP), rabbit anti-Rpl11 antibody (1:1,000, Abcam, ab79352), rabbit anti-Pou5f3 antibody (1:100), mouse anti-GFP antibody (1:1,000, Roche, 11814460001), mouse anti-Ewsr1b antibody (1:100, present study), rabbit anti-Importin β1 antibody (1:1,000, Proteintech, 10077-1-AP), and mouse anti-HuR antibody (1:1000, Santa Cruz Biotechnology; sc-5261).

Techniques: Double Staining, Injection, Immunofluorescence, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Scientific Reports

Article Title: In-frame germline TP53 variant impairs p53 oligomerization and predisposes to cancer

doi: 10.1038/s41598-025-14684-8

Figure Lengend Snippet: Impaired transcriptional activity of the p53 p.E339_F341del isoform. ( A ) Map of the TP53 genetic locus targeted by CRISPR/Cas9. Parental RPE cells were transfected by sgRNA and Cas9, were grown in the presence of nutlin-3 and two clones of RPE-TP53-KO cells were expanded. Genomic DNA was sequenced by NGS. Partial sequence of exon 4 of the TP53 is shown with the target sequence of sgRNA underlined. Note two frameshifting mutations corresponding to the two alleles in RPE-TP53-KO cells. ( B ) Whole cell lysates from parental RPE and RPE-TP53-KO cells incubated or not with nutlin-3 for 12 h were analyzed by immunoblotting. Note induction of p53 and p21 signal after nutlin-3 treatment in parental cells and the absence of p53 and p21 signal in RPE-TP53-KO cells. Staining for importin beta which is an abundant protein involved in nucleocytoplasmic trafficking was used as a loading control . ( C ) Parental RPE and RPE-TP53-KO cells treated with nutlin-3 for 12 h were fixed by PFA, permeabilized by 0.1% TX-100 and analyzed by immunofluorescence microscopy. Representative image is shown. ( D ) Parental RPE, RPE-TP53-KO and RPE-TP53-KO cells stably transfected with wt-p53 (positive control), p53-R248W (negative control) and E339_F341del plasmids were treated with doxycycline and nutlin-3 for 12 h. Whole cell lysates were analyzed by immunoblotting. Staining for importin beta and histone H3 was used as loading controls. ( E ) Parental RPE, RPE-TP53-KO and RPE-TP53-KO cells stably transfected with wt-p53, p53-R248W and E339_F341del plasmids were treated with doxycycline and nutlin-3 for 12 h. After fixation and permeabilisation, cells were probed with p21 and p53 antibodies and analyzed by ScanR microscopy. Mean nuclear intensity of p21 signal was determined in > 300 non-gated RPE and RPE-TP53-KO cells or in the p53-positive RPE-TP53-KO cells rescued by the wild-type or mutant p53. Plotted is the mean ± SD from independent biological replicates (n = 3) normalized to p21 levels in cells expressing the wild type p53. Statistical significance was determined by t-test, ** P < 0.01. ( F ) Parental RPE, RPE-TP53-KO and RPE-TP53-KO cells stably transfected with wt-p53 (positive control), p53-R248W (negative control) and E339_F341del plasmids were treated as in ( E ) and were probed with MDM2 and p53 antibodies. Mean nuclear intensity of MDM2 signal was determined as in ( E ). ( G ) FASAY analysis of the p53-E339_F341del variant transformed into yeast strain yIG397. White colonies (45.3%) contain the functional p53. The fraction of red colonies containing a transcriptionally inactive p53 allele was 54.7%, indicating that the patient is a heterozygote carrying one functional and one transcriptionally inactive p53 allele. Representative image is shown.

Article Snippet: The following antibodies were used: p53 (sc-6243, IF dilution 1:100), p21 (sc-6246, IF dilution 1:100, WB 1:1000), and importin beta (sc-137016) from Santa Cruz; MDM2 (OP46, IF dilution 1:100) from Calbiochem; histone H3 (14269S, WB 1:1000), GAPDH (5174S, WB 1:1000) and p53 (9282S, WB 1:1000) from Cell Signaling Technology; Alexa Fluor-conjugated secondary antibodies (Thermo Scientific).

Techniques: Activity Assay, CRISPR, Transfection, Clone Assay, Sequencing, Incubation, Western Blot, Staining, Control, Immunofluorescence, Microscopy, Stable Transfection, Positive Control, Negative Control, Mutagenesis, Expressing, Variant Assay, Transformation Assay, Functional Assay