Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) TRExRTA BCBL-1 cells bearing the indicated constructs were treated with 25 μM etoposide (Etop; DNA damage agent) for 24 h, 2 μg/mL doxycycline (Dox; viral reactivation) for 24h, or 100 μg/mL cycloheximide (Chx; translational inhibitor) for 6 h, or mock treated with drug diluent and analyzed by western blotting. γH2AX is induced during DNA damage response, KSHV SOX is a viral early protein and marker for infection, and p27 is a short-lived control for translation inhibition. Vinculin is a loading control. Black arrow indicates full length strep-tagged TFIIB and gray arrow indicated full-length endogenous TFIIB. (B) Empty or TFIIB-2xStrep expressing TRExRTA BCBL-1 cells were treated with either 100 ng/mL TRAIL ligand or mock treated with TRAIL dilution buffer for 6 h before western blotting. Caspase-3 cleavage is a control for induction of cell death. (C) Samples in (A) were probed for Caspase-3 and Caspase-8 activation by cleavage. (D) TRExRTA BCBL-1 cells expressing either TFIIB-2xStrep or mutant TFIIB(D207A)-2xStrep were treated with 100 ng/mL TRAIL ligand or mock for 8 h, followed by western blotting. Black arrow indicates cleavage product. (E) TRExRTA BCBL-1 cells expressing TFIIB-2xStrep or mutant TFIIB(D207A)-2xStrep were pretreated with 80 μM of either the caspase-3 inhibitor Z-VAD-DEVD-FMK, the pan-caspase inhibitor Z-VAD-OMe-FMK, or a negative control cysteine protease inhibitor Z-FA-FMK for 2 h. Cells were then treated with either 500 ng/mL TRAIL ligand or mock treated with TRAIL dilution buffer for 7 h followed by western blotting. Boosted strep signal has gamma contrast enhanced for visualization of the cleavage product and GAPDH probed with mouse antibody is a loading control. (F) TFIIB-2xStrep or TFIIB(D207A)-2xStrep expressing TRExRTA BCBL-1 cells were treated with 660 ng/mL TRAIL for 7 h and RNA extracted and sequenced with ribodepletion and ERCC spike-in control RNA for normalization. Volcano plot shows differentially expressed genes in cells containing TFIIB(D207A)-2xStrep relative to cells containing wild-type TFIIB-2xStrep. Genes in red are significantly differentially regulated in TRAIL-treated cells with described links to apoptosis (see Table S1 ), genes in yellow are differentially expressed in both TRAIL-treated and untreated cells, and in blue are genes with unknown function or no known link to apoptosis that are differentially expressed during TRAIL-treatment.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Construct, Western Blot, Marker, Infection, Control, Inhibition, Expressing, Activation Assay, Mutagenesis, Negative Control, Protease Inhibitor

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) TRExRTA BCBL-1 cells treated with 100 μg/mL Chx and endogenous TFIIB protein were analyzed by western blotting. p27 is a control for translational inhibition and GAPDH probed with mouse antibody and vinculin are loading controls. (B) iSLK-KSHV(+) BAC16 cells were reactivated with doxycycline for the given timepoints prior to western blotting. Vinculin serves as a loading control. Note the lack of appreciable procaspase-3 cleavage and activation. These samples come from the same experiment as . (C) iSLK-KSHV(+) BAC16 cells treated with 250 μg/mL cycloheximide (Chx) for given timepoints. Note lack of procaspase-3 activation.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Control, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStrep protein were pulse-labelled for 18 h with L-HPG, harvested at the indicated timepoints post-chase for strep pulldown, then labelled with picolyl-azide-sulfo-Cy5. For quantification, strep signal was normalized to input signal and the 0 h timepoint and plotted. **** P < 0.0001, * P = 0.0103; one-way ANOVA with Tukey multiple comparison test. (B) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStrep were treated with 100 μg/mL cycloheximide (Chx) and harvested at indicated timepoints for western analysis. p27 is a control for Chx activity and vinculin is a loading control. (C) iSLK-KSHV(+) BAC16 cells were treated with 250 μg/mL Chx, harvested at the given timepoints and western blotted for the indicated proteins. Vinculin and GAPDH probed with mouse antibody serve as loading controls. (D) TRExRTA BCBL-1 cells or (E) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStep were treated with 100 μg/mL Chx for 6h with or without 5 μM carfilzomib proteasome inhibitor (carf) and endogenous TFIIB and strep-tagged protein analyzed by western blotting. p53 and p27 are positive controls for Chx activity and FK2 marks poly-ubiquitylated protein as a positive control for proteasomal inhibition; GAPDH probed with mouse antibody is a loading control. (F) TRExRTA BCBL-1 cells treated with 100 μg/mL Chx for 8 h with or without 10 μM TAK-243, a UBA1-targetting inhibitor of ubiquitylation, and western blotted with the indicated antibodies. NRF2 is a control for translation and ubiquitylation inhibition, and GAPDH probed with rabbit antibody is a loading control.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Expressing, Comparison, Western Blot, Control, Activity Assay, Positive Control, Inhibition

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) RT-qPCR of TFIIB RNA normalized to 18S rRNA to confirm TFIIB knockdown in TRExRTA BCBL-1 cells nucleofected with TFIIB siRNA (siTFIIB) or non-targeting siRNA (siNTC) followed by 24 h or 48 h of doxycycline (dox)-induced reactivation or mock. ns-nonsignificant, **** P < 0.0001, *** P = 0.0004, * P = 0.0285; two-way ANOVA with Tukey correction. (B) Western blots of samples in (A). SOX is a viral early gene and K8.1 is a viral late gene. GAPDH probed with mouse antibody is a loading control. (C) PCA analysis of RNA-seq samples described in (A) demonstrating triplicate consistency. (D) Violin plot of gene expression changes for differentially expressed cellular (host) and viral genes from RNA-seq analysis of siTFIIB knockdown relative to siNTC control at 48 h post-reactivation. This is the ensemble of all changes unfiltered by P value. ****P < 0.0001; Student’s T-test with Welch correction. (E) Heatmap of KSHV viral gene expression changes in RNA-seq from siTFIIB and siNTC knockdown at the given timepoints. Cells were reactivated in all conditions and normalized to 24 h gene expression in siNTC cells. (F) Heatmap of viral gene expression changes from RNA-seq analysis of TRExRTA BCBL-1 cells expressing TFIIB-2xStrep or TFIIB(D207A)-2xStrep and reactivated for 48 h.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Control, RNA Sequencing, Gene Expression, Expressing

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) RT-qPCR of TFIIB normalized to 18S RNA harvested from reactivated TRExRTA BCBL-1 cells at the given timepoints. **** P < 000.1, *** P = 0.0008; one-way ANOVA with Tukey multiple comparison test. (B) Western analysis of endogenous TFIIB in TRExRTA BCBL-1 cells or (C) iSLK-KSHV(+) BAC16 cells at timepoints post-reactivation or mock. KSHV SOX is an early gene and vinculin, and GAPDH probed with mouse is a loading control. * P = 0.01636; Student’s t-test. (D) As in (A) but in iSLK-KSHV(+) BAC16 cells. (E) Flow analysis of iSLK-KSHV(+) BAC16 cells transduced with Halo or TFIIB-Halo and mock or 48 h reactivation with doxycycline (dox) and sodium butyrate; Halo was labeled with JF646-Halo dye and measured by signal in the APC-A channel. Fluorescence distributions are shown for a representative replicate and mean APC-A quantification for all samples is shown on the right. ns-nonsignificant, ** P = 0.0057; One-way ANOVA with Tukey multiple comparison test. (F) Confocal microscopy of iSLK-KSHV(+) BAC16 cells transduced with Halo protein or TFIIB-Halo, +/-viral reactivation. Hoescht 3342 delineates nuclei. Scale bar = 20 μm.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Quantitative RT-PCR, Comparison, Western Blot, Control, Transduction, Labeling, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) Western blotting of iSLK cells containing or lacking the KSHV BAC16. Cells were mock treated or dosed with 5 ug/mL doxycycline (Dox) and 1 mM sodium butyrate (NaBut) for 48 h. KSHV SOX is a marker for lytic viral infection and anti-GAPDH probed with rabbit antibody is a loading control. ( B) Flow analysis of iSLK cells expressing TFIIB-Halo and containing or lacking the KSHV BAC16. Cells were treated with Dox and NaBut and harvested 48h later. Halo was labeled with JF646-Halo dye and was measured by the signal in the APC-A channel and the fold change in fluorescence over mock-treated cells was quantified. *P < 0.0307; Student’s unpaired t-test.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Marker, Infection, Control, Expressing, Labeling, Fluorescence

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) iSLK-KSHV(+) BAC16 cells +/-reactivation for 36 h, treated with 250 μg/mL cycloheximide (Chx) for the indicated timepoints, then western blotted with the indicated antibodies. KSHV SOX is a control for infection, p27 is a control for translation inhibition, and GAPDH probed with rabbit antibody is a loading control. (B) TRExRTA BCBL-1 cells or (C) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStrep were mock-treated or reactivated for 18 h, treated with 100 μg/mL Chx, and western blotted for the indicated proteins. Vinculin is a loading control. (D) Western blots of TRExRTA BCBL-1 cells treated with 18 h of TRIM28 or nontargeting control (siNTC) siRNAs, followed by 100 μg/mL Chx treatment for the indicated times. (E) Western blots of TRExRTA BCBL-1 cells +/-18 h reactivation and treatment with 1mM of the viral DNA replication inhibitor phosphonoacetic acid (PAA) followed by a timecourse with 100 μg/mL Chx. Early viral gene SOX expression is not affected by inhibition of DNA replication, but late gene K8.1 is dependent on DNA replication and is thus not expressed. Black arrow indicates appropriate p27 band under non-specific antibody signal. GAPDH probed with mouse antibody is a loading control.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Control, Infection, Inhibition, Expressing

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) Western blots from of TRExRTA BCBL-1 cells nucleofected with TRIM24 siRNA or non-targeting control (siNTC) for 18 h followed by a timecourse with 100 μg/mL cycloheximide treatment (Chx). p27 is a translation inhibitor control and GAPDH probed with mouse antibody a loading control. (B) Western blots of iSLK-KSHV(+) BAC16 cells +/-reactivation and treated with 1mM of phosphonoacetic acid (PAA). SOX is a viral early gene and K8.1 is a late gene (expressed only after DNA replication).

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: Western blot analysis of nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells with an anti-GAPDH mAb ( A ), anti-PCNA mAb ( B ), and anti-N mAb ( C ). ( A,B and C ) Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells. ( C ) Moc, mock-infected cells; Inf, PEDV-infected cells. The arrowheads indicate purified bands that are the same sizes as GAPDH ( A ), PCNA ( B ), and N ( C ) proteins. ( D ) Western blot analysis of N protein in nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells at different times with an anti-GAPDH mAb, anti-PCNA mAb, and anti-N mAb. Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Western Blot, Infection, Purification

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A ) The NPM1 fusion protein as a marker of the nucleolus is colored red. The nucleolar localization of N in transfected cells was clearly observed at 42–42.5 hpt. As shown, a small amount of N was observed in the nucleolus at 42 hpt (t = 30 min). N protein accumulated continuously in the nucleolus of transfected cells until t = 60 min and was exported from the nucleolus at t = 61–65 min. This figure shows snapshots of the cells from the time-lapse movie ( in the ). Data are representative of one of three independent experiments. Real-time visualization of the kinetics of the nucleolar localization of N protein indicated that the process was rapid, taking only 30 min in total. ( B ) Knockdown of NPM1 protein levels following siRNA treatment. Vero E6 cells transfected with no siRNA (Mock), scrambled siRNA (siScr), left untreated (No treat) or with different concentrations (mM) of siRNAs targeting NPM1 (siNPM1) (as indicated at the top of each lane) were harvested 48 hpt. Endogenous NPM1 protein levels were detected by immunoblotting using antibodies directed against the indicated proteins. ( C and D ) Western blot analysis of Myc-N protein in nuclear and cytoplasmic fractions of NPM1-knockdown cells ( C ) or Ectopic NPM1-overexpression cells ( D ) at 48 hpt with anti-GAPDH mAb, anti-PCNA mAb, anti-NPM1 mAb, anti-Myc mAb and anti-Flag mAb. Nuc, nuclear fraction; Cyt, cytoplasmic fraction; cell, whole cells. Densitometric data for Nuc/Cell (Myc-N) from three independent experiments are expressed as mean ± SD.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Marker, Transfection, Knockdown, Western Blot, Over Expression

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A and B ) N protein binding prevents NPM1 proteolytic cleavage. Vero E6 cells were transfected with pMyc-N or empty vector for 24 h and then treated with or without 100 μM of Ac-DEVD-CHO (caspase-3 inhibitor) for 6 h. The cells were treated with or without 250 nm of STS for 18 h. The western blots were probed for freshly extracted proteins with antibodies against NPM1, Myc, GAPDH and caspase-3. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. CF, cleavage fragment. ( C ) N protein enhances the antiapoptotic effect of NPM1. Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h. Genomic DNA was loaded on to a 2% agarose gel. Verification of Myc-N induction and equal sample loading are shown by anti-Myc and anti-GAPDH mAbs. ( D ) Vero E6 cells were transfected with pMyc-N or empty vector for 30 h and then treated with or without 250 nm of STS for 18 h, then TUNEL and DAPI staining to examine the apoptotic cell death. Statistical results represent means ± SD of apoptotic cell counts from six different fields (right).

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Protein Binding, Transfection, Plasmid Preparation, Western Blot, Agarose Gel Electrophoresis, TUNEL Assay, Staining

Journal: Molecular Neurodegeneration

Article Title: Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice

doi: 10.1186/1750-1326-6-87

Figure Lengend Snippet: Golgi fragmentation in Hup23 brainstem . A , Double immunofluorescence analysis of Hup23 brains stained with antibodies against p23 and GM130. Confocal images of neurons in pontine nuclei (upper panels) and a region in the middle of brainstem (lower panels) are shown. Arrowheads indicate abnormal p23 localization and Golgi fragmentation (visualized by GM130 staining) in neurons expressing high levels of p23. B , Immunoblot analysis of UPR markers in brainstem of Hup23 mice. Seventy five μg of total protein lysates of brainstem of Hup23 and non-transgenic littermates were fractionated on 4-20% SDS-PAGE gels and immunoblotted using antibodies against the indicated proteins. Hsc70 and GAPDH levels served as loading controls. Lysates from mouse embryonic fibroblasts (MEF) exposed overnight to 2 μg/ml tunicamycin (Tm) were analyzed on the same gels as positive control for the activation of the UPR.

Article Snippet: The following commercially available antibodies were used in this study - mAb: synaptophysin, MAP2 and GFAP (Sigma), NeuN (Chemicon), MBP (Covance), GM130 (BD Transduction Laboratories), ATF4 and Hsc70 (Santa Cruz Biotechnology), GAPDH (Abcam); polyclonal antibodies: Calbindin-D-28K (Sigma), VGLUT1 and VGLUT2 (Millipore), GFAP (Dako), Iba1 (Wako), and anti αB-crystallin (Serotec).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Transgenic Assay, SDS Page, Positive Control, Activation Assay