rabbit anti crm1  (Novus Biologicals)

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Involvement of the Ran, CRM1, and 14-3-3 proteins in the nuclear exclusion of FOXO1. ( a ) Effects of FOXO1 mutant with the S248A or/and S311A substitution on its combination with 14-3-3 determined by co-immunoprecipitation in presence or absence of 740-Y-P. The chicken GCs were transfected with the expression constructs of the pcDNA3,1(+)-FOXO1 wild-type, pcDNA3,1(+)-FOXO1 Ser248A mutant, and pcDNA3,1(+)-FOXO1 Ser311A mutant, respectively. ( b ) The protein combining level of 14-3-3 with FOXO1 protein in the cultured GCs. ( c ) Effects of FOXO1 mutant with the S248A or/and S311A substitution on its combination with the CRM1 determined by co-immunoprecipitation in presence or absence of 740-Y-P. ( d ) The combining level of CRM1 with FOXO1 protein in the cells. ( e ) Effects of RANQ69L overexpression on the combination of FOXO1 with 14-3-3 or CRM1 examined by co-immunoprecipitation in presence or absence of Ly294002. The chicken GCs were transfected with the pcDNA3,1(+)-FOXO1 expression construct, and simultaneously with the pcDNA3,1(+)- RANQ69L plasmid. ( f ) The protein combining level of 14-3-3 with FOXO1 in the cells under FOXO1 overexpression with or without Ly294002 treatment and with or without RANQ69L treatment by using Western blotting. ( g ) The protein combining level of CRM1 with FOXO1 under the same conditions as ( f ). ( h ). Effects of the two PKB phosphorylation sites in FOXO1 on its combination with RAN. The ovarian GCs were transfected with the expression constructs, pcDNA3,1(+)-FOXO1 wild-type, pcDNA3,1(+)-FOXO1 Ser248A mutant, and pcDNA3,1(+)-FOXO1 mutant, in presence or absence of 740-Y-P or in presence or absence of Ly294002, respectively, which were determined by the co-immunoprecipitation assay. ( i ) The combination levels of Ran with FOXO1 in cultured GCs transfected by the plasmid of pcDNA3,1(+)-FOXO1 Ser248A mutant with/without 740-Y-P treatment or with/without Ly294002 treatment. ( j ) The combination levels of Ran with FOXO1 in the cultured GCs transfected by the plasmid of pcDNA3,1(+)-FOXO1 Ser311A mutant with/without 740-Y-P treatment or with/without Ly294002 treatment. n = 3. ** p < 0.01, * p < 0.05.

Article Snippet: rabbit anti-CRM1 antibody , 1:1000 , Bioss Antibodies, Beijing, China , bs-3145R.

Techniques: Mutagenesis, Immunoprecipitation, Transfection, Expressing, Construct, Cell Culture, Over Expression, Plasmid Preparation, Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: A-E. Reduction of IFIT1 , CXCL10 , ACE2 , TMPRSS2 , and XPO1 mRNA. RT-qPCR analysis of RNA from the experiment shown in Figure 1A ,B . F. 4OI and SEL reduce ACE2 and TMPRSS2 protein levels. Uninfected Calu3 cells were grown in medium containing 4OI (100 µM) or SEL and cellular ACE2 and TMPRSS2 levels were measured by immunoblot after 1 – 72 h. G . 4OI reduces half-life of ACE2. Uninfected Calu3 cells were grown in medium containing 4OI (100 µM) with our without cycloheximide (CHX, 50 µg/ml) and cellular ACE2 levels were measured by immunoblot after 1 – 12 h. H, I. NEDD4L knock-down attenuates the ACE2-destroying capacity of 4OI at the protein (H) and the mRNA level (I). J, K. MDM2 knock-down attenuates the ACE2-destroying capacity of 4OI at the protein (J) and the mRNA level (K). L. 4OI and SEL interfere with cell entry of SARS-CoV-1 and -2. Calu3 cells were pre-incubated with 4OI (48 h) or SEL (24 h or 48 h) and inoculated with luciferase-expressing VSV particles pseudotyped with SARS-CoV-1 or -2 spike protein or VSV-G protein. Cell entry was assessed by luciferase activity 16 h after inoculation. SARS-CoV pseudotype luciferase signal was normalized against the signal obtained with VSV-G pseudotypes, which was set as 100%. Pooled analysis of 6 independent experiments with n =3 replicates each. M. 4OI and SEL reduce XPO1 protein levels. Uninfected Calu3 cells were grown in medium containing 4OI or SEL, and XPO1 levels were measured by immunoblot 1 - 72 h after addition of the compounds. A-E and H: One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (92485S, 1:1000, Cell Signaling Technology), XPO1 (46249S, 1:1000, Cell signaling technology), β-actin (ab49900, 1:20,000, Abcam).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Incubation, Luciferase, Expressing, Activity Assay

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: Experiments for panels A and B were performed in the Gerold lab (Hannover, Germany) and for panels C-G in the Olagnier lab (Aarhus, Denmark). LDH release assay demonstrated that the compounds were non-toxic to Calu3 cells after 48 h of incubation ( Figure S1 ). A,B . Calu3 cells were pretreated with the compounds (BARD, 0.1 µM; SFN, 10 µM; 4OI, 100 µM) for 24 h, inoculated with SARS-CoV-2/München-1.2/2020/984,p3 (MOI = 0.005) in presence of the compounds for 4 h, followed by removing the viral inoculum and adding fresh medium containing the respective compounds and controls. 48 h p.i., viral genome copies were determined by RT-qPCR. A. supernatants and B. cell lysates. C-G . Calu3 cells were pretreated with the indicated compounds for 48 h (C-F) or 24 h (G), infected with SARS-CoV-2 Wuhan-like early European B.1 lineage (FR-4286) (MOI = 0.01) for 1 h, followed by removal of the inoculum and incubation in fresh medium containing the compounds. Target gene expression and protein levels were measured 24 h p.i. C. Reduction of SARS-CoV-2 spike and nucleocapsid proteins, and XPO1 protein expression, but increase in AKR1B10 and NQO1 levels by 4OI (125 µM) (immunoblot with β-actin as internal reference). D. Marked reduction of viral RNA of diverse SARS-CoV-2 variants of concern by 4OI (125 µM). E,F. NRF2 independence of the anti-SARS-CoV-2 effect of 4OI. Control (transfected with control siRNA) or NRF2 knock-down Calu3 cells (transfected with specific anti-NRF2 siRNA) were infected with SARS-CoV-2 (MOI 0.01) and treated with 4OI (125 µM) or buffer only. E . SARS-CoV-2 RNA (RT-qPCR, cell lysates). F. SARS-CoV-2 spike protein (immunoblot with vinculin as internal reference, cell lysates). n =3, means ±SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (92485S, 1:1000, Cell Signaling Technology), XPO1 (46249S, 1:1000, Cell signaling technology), β-actin (ab49900, 1:20,000, Abcam).

Techniques: Lactate Dehydrogenase Assay, Incubation, Quantitative RT-PCR, Infection, Targeted Gene Expression, Expressing, Western Blot, Control, Transfection, Knockdown

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: A-E. Reduction of IFIT1 , CXCL10 , ACE2 , TMPRSS2 , and XPO1 mRNA. RT-qPCR analysis of RNA from the experiment shown in Figure 1A ,B . F. 4OI and SEL reduce ACE2 and TMPRSS2 protein levels. Uninfected Calu3 cells were grown in medium containing 4OI (100 µM) or SEL and cellular ACE2 and TMPRSS2 levels were measured by immunoblot after 1 – 72 h. G . 4OI reduces half-life of ACE2. Uninfected Calu3 cells were grown in medium containing 4OI (100 µM) with our without cycloheximide (CHX, 50 µg/ml) and cellular ACE2 levels were measured by immunoblot after 1 – 12 h. H, I. NEDD4L knock-down attenuates the ACE2-destroying capacity of 4OI at the protein (H) and the mRNA level (I). J, K. MDM2 knock-down attenuates the ACE2-destroying capacity of 4OI at the protein (J) and the mRNA level (K). L. 4OI and SEL interfere with cell entry of SARS-CoV-1 and -2. Calu3 cells were pre-incubated with 4OI (48 h) or SEL (24 h or 48 h) and inoculated with luciferase-expressing VSV particles pseudotyped with SARS-CoV-1 or -2 spike protein or VSV-G protein. Cell entry was assessed by luciferase activity 16 h after inoculation. SARS-CoV pseudotype luciferase signal was normalized against the signal obtained with VSV-G pseudotypes, which was set as 100%. Pooled analysis of 6 independent experiments with n =3 replicates each. M. 4OI and SEL reduce XPO1 protein levels. Uninfected Calu3 cells were grown in medium containing 4OI or SEL, and XPO1 levels were measured by immunoblot 1 - 72 h after addition of the compounds. A-E and H: One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (92485S, 1:1000, Cell Signaling Technology), XPO1 (46249S, 1:1000, Cell signaling technology), β-actin (ab49900, 1:20,000, Abcam).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Incubation, Luciferase, Expressing, Activity Assay

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: A-F . WT and NRF2 −/- human iPSC-derived ECs were infected with the luciferase-labeled strain hCoV-229E-luc (MOI=0.3) for 4 h and then cultured for 48 h in fresh medium containing the compounds. Luciferase activity, viral M protein RNA levels, host mRNA expression, and mitochondrial ROS levels were measured after 48 h. A,B. Luciferase activity and viral M protein RNA. C. ANPEP mRNA. D. XPO1 mRNA. E. Mitochondrial ROS (flow cytometry). F. HMOX1 mRNA. G-I. Effect of HMOX1 knock-down on 229E-luc infectivity and antiviral activity of the compounds in A549 cells. Infections and treatments were carried out as in A-F, except that WT (transfected with scrambled control siRNA) or siRNA-mediated HMOX1 knock-down A549 cells were used. G. Luciferase activity. H. M protein RNA. I . ANPEP mRNA. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (92485S, 1:1000, Cell Signaling Technology), XPO1 (46249S, 1:1000, Cell signaling technology), β-actin (ab49900, 1:20,000, Abcam).

Techniques: Derivative Assay, Infection, Luciferase, Labeling, Cell Culture, Activity Assay, Expressing, Flow Cytometry, Knockdown, Transfection, Control

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: XPO1 mRNA was knocked down in A549 cells by siRNA, and infectivity of 229E-luc and antiviral efficacy of the compounds were assessed in WT (transfected with scrambled control siRNA) and XPO1 KD cells following the same infection protocol as in . A. XPO1 mRNA (RT-qPCR). B. Luciferase activity. C. M protein RNA expression. D. ANPEP mRNA (RT-qPCR). E. NFE2L2 mRNA (RT-qPCR). F. HMOX1 mRNA (RT-qPCR). n =3. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (92485S, 1:1000, Cell Signaling Technology), XPO1 (46249S, 1:1000, Cell signaling technology), β-actin (ab49900, 1:20,000, Abcam).

Techniques: Infection, Transfection, Control, Quantitative RT-PCR, Luciferase, Activity Assay, RNA Expression

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: 4OI reduces transcription of the XPO1 gene. A. KEGG pathway analysis based on XPO1 cargoes listed in the ValidNESs database . B. Efficiency of ABCB1 mRNA knock-down with siRNA (RT-qPCR). C. Comparison of half-life reduction of XPO1 mRNA by ActD, 4OI, or co-treatment with both. Numerical values for half-life were obtained by extrapolation and are listed in the table next to the graph. D. Same experiment as in C but using ABCB1 knock-down (siRNA) A549 cells. Representative of two independent experiments, n =3. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (92485S, 1:1000, Cell Signaling Technology), XPO1 (46249S, 1:1000, Cell signaling technology), β-actin (ab49900, 1:20,000, Abcam).

Techniques: Knockdown, Quantitative RT-PCR, Comparison