coding sequence of the eegf  (GenScript corporation)

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Silencing of GmMEKK2 by virus‐induced gene silencing (VIGS) increased soybean mosaic virus (SMV) susceptibility. (A) Efficiency of GmMEKK2 silencing in empty vector control (EV) and GmMEKK2 ‐silenced mekk2 i1 and mekk2 i2 plants at 0, 7, 14 and 21 days post‐inoculation (dpi). (B) Phenotypes of soybean after SMV infection: EV and GmMEKK2 ‐silenced lines generated using VIGS. Images were taken at 21 dpi. (C) Disease indices of plants at 21 dpi. Lowercase letters denote statistically significant differences among groups at the same time point ( p < 0.05, one‐way ANOVA with Duncan's test). (D) Relative SMV accumulation in top new leaves of EV and GmMEKK2 ‐silenced plants at 7, 14 and 21 dpi, quantified by reverse transcription‐quantitative PCR using SMV coat protein‐specific primers.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Virus, Plasmid Preparation, Control, Infection, Generated, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Overexpression of GmMEKK2 improved soybean mosaic virus (SMV) resistance in soybean. (A) Infection symptoms on soybean leaves after SMV inoculation. NT, nontransgenic plants; ZMP1, 3, 6 and 7 indicate GmMEKK2 ‐overexpression lines 1, 3, 6 and 7, respectively. (B) Disease indices of NT and each GmMEKK2 ‐overexpression line. The disease index was investigated at 21 days post‐SMV‐inoculation. (C) Quantification of SMV content in soybean leaves. SMV‐susceptible line 1138‐2 was used as a positive control. (D) The GmMEKK2 expression pattern in NT plants after SMV inoculation. (E) Comparison of yield traits between NT and overexpression plants after SMV infection. Mock‐inoculated plants served as the control. Values labelled with different lowercase letters (a–e) are significantly different at p < 0.05 as determined by Duncan's test.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Over Expression, Virus, Infection, Positive Control, Expressing, Comparison, Control

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Expression profiles of key differentially expressed genes (DEGs) between nontransgenic (NT) and GmMEKK2 ‐overexpression lines (ZMP) involved in the reactive oxygen species (ROS)‐ and salicylic acid (SA)‐related pathways. (A) KEGG enrichment analysis of DEGs between NT and ZMP plants. Left: NT_CK versus ZMP_CK (uninfected controls); Right: NT_7d versus ZMP_7d (7 days post‐SMV‐inoculation [dpi]). Points represent enriched pathways, with size indicating gene count and colour reflecting −log 10 (adjusted p ‐value). Red arrows highlight defence‐related pathways. (B) Expression dynamics of key components among MAPK, plant hormone signalling and plant–pathogen interaction pathways. Schematic depicts signal transduction from apoplast to cytoplasm, including Ca 2+ sensors (CNGCs and CDPKs), ROS producers (Rbohs) and SA‐induced defence protein (PR1). Heatmaps show expression levels across conditions (NT and ZMP at 0, 7 and 14 dpi), with gene IDs labelled.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Expressing, Over Expression, Transduction

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Kinase activity of GmMEKK2 is dispensable for its function in mediating defence signalling. (A–E) Relative expression levels of (A) GmMKK1 , (B) GmMPK4A , (C) GmMPK13‐like , (D) GmSUMM2 and (E) GmCRCK3 in nontransgenic control (NT), GmMEKK2 ‐overexpression lines (ZMP1, ZMP3 and ZMP7), empty vector control (EV) and GmMEKK2‐ silenced lines ( mekk2 i1 and mekk2 i2 ). Lowercase letters denote significant differences at p < 0.05 as determined by one‐way ANOVA with Duncan's test. (F) Domain architecture of GmMEKK2 highlighting the kinase domain (6–264 amino acids) and ATP‐binding site (K36). Autophosphorylation of GmMEKK2 was assessed by immunoblotting with α‐pSer/Thr antibody. Recombinant proteins GmMEKK1‐FLAG and GmMEKK1 K321M ‐FLAG were used as positive and negative controls, respectively. Coomassie brilliant blue staining validated the equal loading of recombinant proteins. (G) Yeast two‐hybrid analysis of GmMEKK2 interaction with GmMKK1, GmMPK4A and GmMPK13‐like. Transformants expressing pGADT7 and pGBKT7 constructs were grown on SD/−Leu/−Trp (control) and SD/−Leu/−Trp/−Ade/−His (selection) media. (H–J) Glutathione S‐transferase (GST) pull‐down assays with anti‐His and anti‐GST antibodies demonstrating direct binding between GST‐GmMEKK2 and (H) GmMKK1‐His, (I) GmMPK4A‐His and (J) GmMPK13‐like‐His.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Activity Assay, Expressing, Control, Over Expression, Plasmid Preparation, Binding Assay, Western Blot, Recombinant, Staining, Construct, Selection

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: GmMEKK2 promotes the immune response induced by salicylic acid (SA). (A) Contents of free (SA) and bound salicylic acid (SAG) in nontransgenic (NT) and GmMEKK2 ‐overexpression (ZMP) lines. (B) GmMEKK2 expression in NT plants after exogenous hormone treatments. ETH, ethylene; ABA, abscisic acid (C–H) Expression of pivotal genes in the SA signalling pathway in NT, GmMEKK2 ‐overexpression and GmMEKK2 ‐silenced ( mekk2 i1 and mekk2 i2 ) plants at 7 days post‐inoculation. EV, empty vector. Values labelled with different lowercase letters (a–c) are significantly different at p < 0.05 as determined by Duncan's test.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Over Expression, Expressing, Plasmid Preparation

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: GmMEKK2 is involved in the regulation of reactive oxygen species homeostasis in soybean. (A, B) H 2 O 2 and O 2− levels in leaves were detected at 7 days post‐inoculation (dpi) using 3,3′‐diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively. The mock‐inoculated leaves were sampled as controls. (C–G) Trends in the gene expression of antioxidases were measured after soybean mosaic virus (SMV) infection. CK, noninoculated control (H–J) Antioxidase activities were measured. POD, peroxidase; CAT, catalase; SOD, superoxide dismutase. The statistical analysis was independently performed for GmMEKK2 ‐overexpression lines ZMP1, ZMP3 and ZMP7, and gene‐silenced lines mekk2 i1 , mekk2 i2 and nontransgenic (NT) plants at each stage. Values labelled with different lowercase letters are significantly different at p < 0.05 as determined by Duncan's test.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Staining, Gene Expression, Virus, Infection, Control, Over Expression

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Molecular mechanisms underlying the GmMEKK2‐mediated regulation of soybean mosaic virus (SMV) resistance in soybean. (A) Phenotype and regulatory mechanism of GmMEKK2 ‐overexpression plants under SMV inoculation. Left: GmMEKK2 ‐overexpression plants (ZMP) show no visible SMV symptoms with autoimmunity phenotype such as leaf yellowing. Right: In ZMP plants, GmMEKK2 (orange ellipses) interacts with GmMKK1 and GmMPK4A, blocking the phosphorylation (letter P in a blue circle) of the GmMEKK1‐GmMKK1‐GmMPK4A cascade. This inhibition represses (cross in a red circle) WRKY transcription factors and leads to non‐phosphorylated CRCK3 releasing SUMM2. This then triggers defence responses such as salicylic acid (SA)‐induced gene expression and basal reactive oxygen species (ROS) accumulation. The elevated ROS constitutivly results in autoimmunity in ZMP plants. (B) Left: Nontransgenic (NT) plants exhibit severe SMV symptoms such as mosaic leaves and mottled pods. Right: In NT plants, GmMEKK2 expression is low, so the GmMEKK1‐GmMKK1‐GmMPK4A cascade remains active. GmMPK4A phosphorylates CRCK3, which binds with and represses SUMM2. This suppresses defence responses, and leads to a ROS burst.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Virus, Over Expression, Blocking Assay, Phospho-proteomics, Inhibition, Gene Expression, Expressing

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: a . Diagram of key molecules in the chemotaxis network. Extracellular ligands (black semicircles) bind to receptors and modulate the activity of CheA, the kinase that phosphorylates the diffusible messenger CheY. CheA activity adaptation is mediated by the methylase CheR and de-methylase CheB. CheY-P binds to the C-ring and triggers a conformational change that results in BFM changing direction (CCW → CW). The swimming bacterium switches from a smooth run to a tumble. b . AlphaFold structure predictions of chimeric molecules. Grey is E. coli CheY, blue is cpAsLOV2, black is a short linker, and orange is the FliM peptide. Cartoons depicting the “caged” dark-state of Opto-CheY and the constitutively active control are shown with the same color code. c . Blue-light absorption triggers unfolding of the J α helix, and CheY activation by removal of the FliM plug from the CheY active site. Active CheY binds to the C-ring and promotes the rotational switch of the BFM from CCW → CW. Light pulses (blue dots, ~100 µ J/cm 2 ) are applied every 6 seconds while monitoring BFM rotational state. Each response is a CW rotation (vertical black line) following a blue light pulse (blue dot) in an otherwise mostly CCW motor (bottom trace)

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Chemotaxis Assay, Activity Assay, Control, Activation Assay

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet:

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Transformation Assay, Expressing, Variant Assay

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Blue light pulses (2 msec, ~100 µ J/cm 2 ) are applied every 6 seconds. Results are shown from 3 different strains: 657 (no plasmid), 638 , and 658 (CheY-cpAsLOV2 N449S ). Binary traces of cell body rotation (CCW=0, CW=1) are shown as a running average of 100 frames (500 msec). Blue light pulses are shown as vertical blue lines.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Averaged binary traces (100 frames running average, 500 msec) for strain 636 (Opto-CheY V416T ). Each plot is a single motor trace. Blue light pulses are shown as blue lines. 2 msec blue light pulses of a given blue light intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}) are applied at constant frequency: every 4 sec for x = 1, every 6 sec for x = 2, every 8 sec for x = 4, every 10 sec for x = 8, every 12 sec for x = 16, and every 15 sec for x = 32.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques:

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: a . Opto-CheY V416T -driven motor responses. Each motor is flashed with a series of pulses of increasing relative intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}, I min delivers ~ 7 · 10 12 blue photons/cm 2 ). Response probability for each BFM at a given intensity is calculated as the number of responses divided by the number of flashes. P see with θ = 2 is used to fit each dose response curve . Ten individual BFM response curves and fits are shown translated horizontally relative to half-maximum response intensity. b . The dose response of one Opto-CheY V416T motor is shown along with best fit curves for fixed values of θ : θ = 2 (black line), θ = 1 (shallower dashed black line), θ = 3 (steeper dashed black line). c . A histogram of most probable θ for 20 individual BFM response curves, corresponding to the fit with highest R 2 .

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques:

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Opto-CheY V416T , Opto-CheY N449S , and Opto-Che single motor dose response curves, as indicated. Each motor responds to light pulses of increasing relative intensity ( I = I min · x, x = {1, 2, 4, 8, 16, 32}) controlled using neutral density filters. Response probability for each BFM expressing an Opto-CheY variant at a given blue light intensity is calculated as the number of responses divided by the number of flashes. P see with θ = 2 fits are shown for each of the 20 Opto-CheY V416T dose response measurements in corresponding color. R 2 values for the fits are given in . For Opto-CheY N449S strains, data is fitted to b× P see , where b is between 0 and 1, a free parameter to allow response probability saturation at values less than 1.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Expressing, Variant Assay

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Averaged binary traces (100 frames running average, 500 msec) are shown for strain 630 . Tethered cells (Opto-CheY N449S ) are subjected to 2 msec pulses every 6 sec at the highest intensity (vertical blue lines). Following are 20 sec pulses (horizontal blue lines) at 1 / 32 of the maximum intensity.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques:

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: AlphaFold first ranked model predictions are shown for Opto-CheY N449S and Opto-CheY V416T , color scheme as in . Red arrow heads point to the location of the introduced mutations. Below, the cryo-EM structure of the flagellar hook from Salmonella is shown (pdb ID 7CGB). One FlgE subunit of the hook is highlighted in blue. AlphaFold structure prediction of FlgE AviRRR is shown to the right (engineered loop in red).

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Cryo-EM Sample Prep

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) Number of peptides corresponding to importin β family proteins identified by MS. Immunoprecipitation and sample preparation from HeLa S3 cells expressing traptamer FA-JX4 or control FA were performed as described in fig. S1B. All MS data are in table S1. ( B ) HeLa cells were infected with HPV16.L2F (MOI, ~0.4; GFP reporter plasmid) and treated at infection with 5 to 10 μM importazole (IPZ) or DMSO. At 48 hpi, GFP-positive cells were quantified by flow cytometry and normalized to DMSO (0 μM IPZ) controls. Data ( n = 3) are shown as individual points, means, and SDs; statistical significance was determined versus DMSO-treated cells by two-tailed, unequal variance t test. ** P < 0.01 and *** P < 0.001. ( C ) HeLa cells were transfected with 10 nM of the indicated siRNA for 48 hours and then infected (MOI, ~0.2) with HPV16.L2F (GFP reporter plasmid). At 48 hpi, GFP-positive cells were measured by flow cytometry and normalized to Scr siRNA controls. Data ( n = 3) are analyzed and presented as in (B), with statistical significance determined versus Scr-treated cells. * P < 0.05 and ** P < 0.01. ( D ) HeLa cells were transfected with siRNA-resistant HA-IPO7-mCherry or control HA-mCherry for 24 hours and then with Scr or IPO7 siRNA #2 for 48 hours and infected (MOI, ~0.2) with HPV16.L2F (GFP reporter plasmid). At 48 hpi, GFP expression in mCherry-positive cells was quantified by flow cytometry and normalized to the Scr siRNA + HA-mCherry control. Data ( n = 3) are analyzed and presented as in (B), with statistical significance determined between samples. * P < 0.05 and *** P < 0.001. ( E ) As in (C), except HaCaT cells were analyzed. *** P < 0.001. ( F ) As in (C), except HPV5.L2F (MOI, ~0.4) was used. *** P < 0.001. ( G ) As in (C), except HPV18.L2F (MOI, ~0.4) was used. *** P < 0.001.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Immunoprecipitation, Sample Prep, Expressing, Control, Infection, Plasmid Preparation, Flow Cytometry, Two Tailed Test, Transfection

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) HeLa S3 cells were transfected with 10 nM Scr or IPO7 siRNA #2 for 48 hours and then left uninfected or infected with HPV16.L2F (MOI, ~100). At 32 hpi, PLA (signals shown in green) was performed with antibodies recognizing FLAG (to detect L2) and GM130. Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( B ) PLA fluorescence intensity per cell (>100 cells) as in (A), shown as individual values, means, and SDs, with statistical significance determined by one-way analysis of variance (ANOVA). *** P < 0.001 and **** P < 0.0001. ( C ) As in (A), except infection at MOI of ~150 with HPV16.L2F-containing EdU-labeled genome. Cells were fixed 32 hpi, subjected to Click-iT EdU detection (green), and immunostained for Nesprin-2 (magenta). Scale bar, 10 μm. ( D ) Nuclear, extranuclear, and total EdU intensity per cell in multiple images as in (C). Using a cutoff defined as the mean intensity per uninfected cell plus one SD, all 89 control cells and 86 of 89 IPO7 KD cells were EdU positive. One-way ANOVA was used to determine statistical significance. ns, not significant; **** P < 0.0001. ( E ) As in (A), except infection at MOI of ~15. Cells were fixed at 32 hpi and immunostained for FLAG (green) and GM130 (magenta); DNA was stained by DAPI (blue). Cells displaying condensed chromosomes in DAPI staining and fragmented Golgi (GM130 puncta) were identified as mitotic cells. Similar results were obtained in two independent experiments. Scale bars, 10 μm. ( F ) Ratio of FLAG intensity on condensed chromosomes to total per mitotic cell (>27 mitotic cells) as in (E), shown as individual values, means, and SDs, with statistical significance determined by two-tailed, unequal variance t test. **** P < 0.0001.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Transfection, Infection, Staining, Fluorescence, Labeling, Control, Two Tailed Test

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) HeLa cells were mechanically homogenized and fractionated through a 0.5 to 1.6 M sucrose gradient. Portions of each fraction (#1 to #11, top to bottom) were immunoblotted with the indicated antibodies to identify organelle markers. Fractions #3 containing TGN46 and GM130 represents the Golgi-enriched fraction. ( B ) Fraction #3 collected from (A) was subjected to IP with an anti-GM130 antibody or a control IgG and then immunoblotted with the indicated antibodies. ( C ) HeLa S3 cells were transfected with 10 nM Scr or IPO7 siRNA #2 for 48 hours and then uninfected or infected with HPV16.L2F (MOI, ~100). PLA (green) for IPO7 and GM130 was performed at 30 hpi; nuclei were stained with DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( D ) PLA fluorescence intensity per cell (>80 cells) as in (C), shown as individual values, means, and SDs, with statistical significance determined by one-way ANOVA. *** P < 0.001 and **** P < 0.0001. ( E ) Uninfected HeLa cells were transfected with the mNG2-SREBP1 reporter construct for 48 hours and then with 10 nM Scr or IPO7 siRNA #2 for another 48 hours. Samples were fixed and permeabilized, and nuclei were stained with DAPI. The nuclear import of mNG2-SREBP1 was indicated by colocalization of mNG2-SREBP1 fluorescence (green) and DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( F ) Ratio of nuclear to total mNG2-SREBP1 intensity per cell (>26 mNG-positive cells) as in (E) is plotted as individual values, means, and SDs, with statistical significance determined by two-tailed, unequal variance t test. **** P < 0.0001.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Control, Transfection, Infection, Staining, Fluorescence, Construct, Two Tailed Test

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) HeLa S3 cells were uninfected or infected with HPV16.L2F (MOI, ~100). PLA (green) for IPO7 and FLAG (to detect L2) was performed at 24 hpi; nuclei were stained with DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( B ) PLA fluorescence intensity per cell (>100 cells) as in (A), shown as individual values, means, and SDs, with statistical significance determined by one-way ANOVA. **** P < 0.0001. ( C ) Whole-cell extracts from uninfected or HPV16.L2F-infected (MOI, ~1) HeLa cells were collected at the indicated time points. A portion of the resulting extracts (input) was analyzed by immunoblotting for FLAG (to detect L2) and IPO7. The remaining extracts were subjected to IP with an anti-IPO7 antibody or control IgG and analyzed by immunoblotting for FLAG and IPO7. ( D ) HeLa cells were pretreated with 9 μM RO3306 or DMSO as a control for 24 hours and then uninfected or infected with HPV16.L2F (MOI, ~1), with RO3306 or DMSO maintained in the medium. At 24 hpi, cells were lysed and analyzed in (C). ( E ) HeLa cells treated with 10 nM Scr or a mixture of 5 nM COPA and 5 nM COPG1 ( COPA/G1 ) siRNA were uninfected or infected with HPV16.L2F PsV (MOI, ~1). At 24 hpi, cells were lysed, and a portion of the extracts (input) was immunoblotted for FLAG, IPO7, γ-COP, or β-actin (as a loading control). The remaining extracts were subjected to IP with an anti-IPO7 antibody or a control IgG and analyzed as in (C). ( F ) As in (E), except cells were transfected with 10 nM Scr or IPO7 siRNA #2 before infection. Samples were subjected to IP with an anti–γ-COP antibody or control IgG and analyzed as in (E).

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Infection, Staining, Fluorescence, Western Blot, Control, Transfection

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) Diagram of the NLSs in HPV16 L2. Three postulated NLSs are indicated as red boxes, with basic amino acids in the NLSs highlighted in red. The predicted transmembrane domain (TMD) spanning amino acids 45 to 67 is denoted as a black box. The RBS is indicated as a blue box, the amino acids required for retromer binding are highlighted in blue, and the corresponding RBS mutation to alanines are underlined in the DM peptide. The cNLS mutation to alanines are underlined in the 6A peptide. ( B ) Whole-cell extracts of uninfected HeLa cells were incubated with N-terminal biotin-tagged peptides containing residues S296–S316 or S434–R461 of HPV16 L2. The pull-down experiment without peptide was used as a negative control. Samples captured with streptavidin beads were analyzed by immunoblotting for IPO7 or β-actin. ( C ) Coomassie stain of the purified proteins. EGFP-FLAG, C-terminal FLAG-tagged EGFP. FLAG-IPO7, N-terminal FLAG-tagged IPO7. ( D ) N-terminal biotin-tagged peptides containing residues S434–R461 of HPV16 L2 (WT) and the corresponding DM and 6A mutant peptides as indicated in (A) were incubated with purified EGFP-FLAG or FLAG-IPO7. Samples captured with streptavidin beads were analyzed by Western blotting (WB) for FLAG. ( E ) HeLa cells were transfected with indicated DNA constructs for 24 hours to express WT HA-(Δ1–67) L2-3xFLAG (WT) or 6A HA-(Δ1–67) L2-3xFLAG (6A). The cells were lysed, and the resulting extracts were analyzed as in . ( F ) HeLa cells treated with 20 nM Scr or KPNA2 siRNA were uninfected or infected with HPV16.L2F PsV (MOI, ~1). At 24 hpi, cells were lysed and analyzed as in , except antibodies against FLAG, IPO7, KPNA2, or HSP90 (as a loading control) were used to analyze the input.

Article Snippet: The IPO7 coding sequence was amplified from pUC19-mIPO7 (MG5A1798-U, Sino Biological) and cloned into the pCMVTNT-HA-COPG1-mCherry plasmid ( ) to replace COPG1 .

Techniques: Binding Assay, Mutagenesis, Incubation, Negative Control, Western Blot, Staining, Purification, Transfection, Construct, Infection, Control