anti p24 hiv 1  (Polymun Scientific)

Journal: Cell Insight

Article Title: Molecular basis of fox ACE2 recognition by receptor binding domains of SARS-CoV-2 and PCoV-GD

doi: 10.1016/j.cellin.2026.100314

Figure Lengend Snippet: Pseudovirus entry and cell membrane fusion mediated by SARS-CoV-2 and PCoV-GD spikes. (a) Western blotting detection of SARS-CoV-2 and PCoV-GD spike proteins in pseudovirus packaging cell lysate and pseudovirions. The SARS-CoV-2 and PCoV-GD spikes contain a C-terminal Strep-II tag and are detected using anti-Strep II antibody. For cell lysate, β-Actin is used as cellular internal control. For pseudovirions, HIV-1 p24 is used as viral internal control. (b) Western blotting detection of fACE2 and hACE2 protein in stable cell lines. The ACE2s contain a C-terminal Strep II tag and are detected using anti-Strep II antibody. β-Actin is used as cellular internal control. (c) The pseudovirus entry efficiency of SARS-CoV-2 and PCoV-GD into HEK293T, hACE2/HEK293T and fACE2/HEK293T cells. Pseudovirus without spike protein is used as negative control. Statistical comparisons are made using t -test. ∗∗∗, p < 0.001, ns: not significant. Fluorescence intensities are presented as mean ± SEM of three biological replicates. (d) Cell membrane fusion activity mediated by SARS-CoV-2 and PCoV-GD spikes assessed by DSP assay. Cells transfected with DSP 1-7 or DSP 8-11 are used as negative control. Fluorescence images are acquired after 8 h of co-incubation. Scale bars: 50 μm.

Article Snippet: After blocked with 5% (w/v) fat-free milk in tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 (TBST) at room temperature for 2 h, the membranes were incubated with primary antibodies against β-actin (Cat: LF201S, Epizyme Biotech), HIV-1 p24 protein (Cat: 11695-R002, SinoBiological) or the Strep II tag (Cat: AE066, Abclonal) in Protein-Free Blocking and Antibody Dilution Buffer (Beyotime Biotechnology) for 2 h. After washing in TBST buffer for three times, the membranes were then incubated with HRP-conjugated secondary antibodies (Abclonal) for 2h and later detected by a ChemiDoc MP Imaging System (Bio-Rad).

Techniques: Membrane, Western Blot, Control, Stable Transfection, Negative Control, Fluorescence, Activity Assay, Transfection, Incubation

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative transmission electron micrographs of gp120-VLP particles. Original scale bar 100 nm. ( B ) Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). ( C ) Flow cytometry analysis of VLPs (Env(-) and expressing x4-gp120) bound to latex beads in the presence of soluble human CD4, using an anti-Histidine mAb. A representative experiment is shown of 3 performed. ( D ) Representative transduction experiments using the indicated LVPs, where the reporter expression was captured using the Tecan SparkCyto reader. Upper panels show bright-field images of target HEK-293 CD4 cells. Lower panels show fluorescence signal from GFP expression in transduced cells. Images were captured with a 4× objective, using an exposure of 200ms in all cases and 80ms for control VSVG-VLPs (positive control). Env(-) LVPs were used as negative control (n=3). ( E ) Quantification of the Mean Fluorescence Intensity (MFI) of images obtained in transduction experiments. Figure 2—figure supplement 1—source data 1. Original files for western blot analysis for . Figure 2—figure supplement 1—source data 2. PDF file containing original western blots for . Original membranes corresponding to , panel B. Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). , panel B shows the last six lanes of these membranes reorganized to separate LVPs from VLPs. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Transmission Assay, Transfection, Construct, Generated, Western Blot, Molecular Weight, Flow Cytometry, Expressing, Transduction, Fluorescence, Control, Positive Control, Negative Control

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative images of clarified VLPs visualized by STED microscopy. Upper panels show images of the indicated VLPs stained for Gag p24 (blue) and gp120 (red). Lower panels show ×10 magnification of equivalent images. White arrows indicate mature VLPs (p24 condensation). ( B ) Percentage of mature VLPs, analyzed from the images in ( A ) using TrackAnalyzer in ImageJ, based on p24 intensity and aggregation level (mean ± SD; n=2; ****p≤0.0001; the significance indicated on immature VLPs bar shows the difference with all other conditions). ( C ) Percentage of VLPs expressing gp120 on their surface, as analyzed in ImageJ (mean ± SD; n=2; ***p≤0.001). ( D ) Distribution of gp120 mean fluorescence intensity. Each spot corresponds to the mean fluorescence intensity for each analyzed VLP in a.u. The black line represents the mean of all values (****p≤0.0001). ( E ) Frequency of gp120 intensity/particle. Statistical significance was determined by one-way-ANOVA followed by Tukey’s multiple comparisons test in panels B and C and by Mann-Whitney analysis for panel D.

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Microscopy, Staining, Expressing, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: The presence of CXCR4 R334X on JKCD4 + cells does not alter gp120 binding and increases fusion events with target cells expressing HIV pHXB2 envelope. ( A ) Binding of X4-gp120 to target cells expressing CD4 and CXCR4 or CD4 and CXCR4 R334X analyzed by flow cytometry. Cells were incubated with 0.3 mg/mL of X4-gp120 at 37 °C for 30 min. Data show MFI (arbitrary units, a.u.) mean ± SD; (n=2). Statistical significance was determined using Student’s t-test (n.s.=not significant). ( B ) Cell-cell fusion between JKHXBc2-expressing HIV-1 envelope and different target cells (JKCD4 + CXCR4 + , JKCD4 + CXCR4 - , and JKCD4 + CXCR4 R334X ). Prior to co-culture, each cell type was loaded with the corresponding cell-tracker. Data show the percentage of fusion events ± SD (n=6). We used as reference the fusions events detected in JKCD4 + CXCR4 + cells (100%). Statistical significance was determined by one-way-ANOVA (*p<0.05, ****p≤0.0001). ( C ) Representative biparametric histograms from cells in B showing CMAC versus orange fluorophores. ( D ) Human PBMCs isolated from a WHIM patient (WHIM) and three healthy donors (HD1-3) in two independent experiments were infected with X4-pseudotyped HIV-1 NL4-3 (MOI: 0.001). At 2 hr post infection (p.i.), supernatant samples were obtained at different time points (days post-infection) and p24 levels (pg/mL) in each sample were determined using a commercial ELISA. Results show mean ± SD (n=2).

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Co-Culture Assay, Isolation, Infection, Enzyme-linked Immunosorbent Assay

Journal: Virulence

Article Title: HIV-1 Vpr activates microglia by upregulating m6A modification through ubiquitin-proteasome pathway-mediated degradation of the demethylase ALKBH5

doi: 10.1080/21505594.2026.2645908

Figure Lengend Snippet: Identification of viral proteins on up-regulating of m6A modification in microglia. HMC3 cells were transfected with Flag-fused viral proteins expression plasmids for 24 h, the relative m6A modification levels were measured by the dot blot assay using an anti-m6A antibody, and MB staining was used as a loading control (A). The expression of viral proteins was detected with anti-Flag antibody by western blotting, and ACTIN was used as the loading control (the asterisk indicated the viral protein) (B). HMC3 cells were transfected with the increasing concentration of HA-Vpr plasmid for 24 h, the relative m6A levels were measured by the dot-blot assay (C) and m6A ELISA (D), and the expression of Vpr was detected with anti-HA antibody by western blotting (E). HMC3 cells were infected with HIV-1 or HIV-1-ΔVpr at 100 ng of p24/ml for 48 h, the relative m6A levels were measured by the dot-blot assay (F) and m6A ELISA (G), the expression of Pr55-Gag and Vpr were detected with anti-P24 and anti-Vpr antibodies respectively by western blotting (H). Statistical significance was analyzed using a t -test. Asterisks indicate significant differences (***, p < 0.001; ****, p < 0.0001).

Article Snippet: P24 , sc-69728 , , Santa Cruz Biotechnology.

Techniques: Modification, Transfection, Expressing, Dot Blot, Staining, Control, Western Blot, Concentration Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Infection

Journal: Virulence

Article Title: HIV-1 Vpr activates microglia by upregulating m6A modification through ubiquitin-proteasome pathway-mediated degradation of the demethylase ALKBH5

doi: 10.1080/21505594.2026.2645908

Figure Lengend Snippet: The effects of Vpr on the expression of ALKBH5. HMC3 cells were infected with or without HIV-1 at 100 ng of p24/ml for 48 h. The protein levels of METTL3, METTL14, WTAP, FTO, ALKBH5, and Pr55-Gag were detected with corresponding antibodies respectively by western blotting, and GAPDH was used as the loading control, and the relative protein levels were quantified with ImageJ software (A). The mRNA levels of enzymes were detected by RT-qPCR (B). HMC3 cells were transfected with different concentrations of HA-Vpr plasmid for 24 h. The above protein levels were detected with corresponding antibodies, and the relative protein levels of ALKBH5 were quantified (C). HMC3 cells were infected with HIV-1 or HIV-1-ΔVpr at 100 ng of p24/ml for 48 h. The protein levels of ALKBH5, Pr55-Gag, and Vpr were detected with corresponding antibodies respectively, and the relative protein levels of ALKBH5 were quantified (D). Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01).

Article Snippet: P24 , sc-69728 , , Santa Cruz Biotechnology.

Techniques: Expressing, Infection, Western Blot, Control, Software, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: HIV-1 Vpr counteracts TASOR restriction to promote infection prior to integration

doi: 10.64898/2026.03.06.710055

Figure Lengend Snippet: HIV-1 Vpr overcomes TASOR mediated restriction and promotes infection in MDMs. a , Western blotting of TASOR, REAF and phosphorylated histone H3 (pH3 Ser10/Thr11) in Jurkat cells treated for 24 hours with HIV-1 Vpr or SIVmac239 Vpx VLPs (50ng measured by p24 ELISA). b , Western blotting of REAF, TASOR, MUS81 and GFP in HeLa and HeLa-ΔTASOR. c , Infectivity (FFU/ml) of HIV-1 89.6 WT on HeLa-CD4 (∅), HeLa-ΔTASOR-CD4 and HeLa-ΔPPHN1-CD4. Error bars indicate standard deviations of the means derived from duplicate titrations. Ordinary one-way ANOVA with multiple comparisons and Dunnet’s test. d , HeLa-CD4 and HeLa-ΔTASOR-CD4 challenged with HIV-1 89.6 WT , HIV-1 89.6 Δ vpr , HIV-1 89.6 Q65R or HIV-1 89.6 F34I . Fold increase in infectivity measured by ELISA of p24 protein in cell culture supernatant. Error bars represent standard deviations from the means of replicates. Ordinary one-way ANOVA with multiple comparisons and Holm-Šídák correction. e , Infectivity (FFU/ml) of HIV-1 2044 on HeLa-shTASOR-CD4 and HeLa-shREAF-CD4. Error bars represent standard deviations from the means of titrations performed in duplicate. Ordinary one-way ANOVA with multiple comparisons and Dunnet’s test. f , Fold increase in infectivity (FFU/ml) of HIV-1 89.6 WT , HIV-1 89.6 Δ vpr and HIV-1 89.6 Δ vpu on HeLa-CD4 cells with shRNA targeting TASOR/REAF compared to control shRNA cells. Error bars represent standard deviations from the means of titrations performed in duplicate. Two-way ANOVA with multiple comparisons and Dunnet’s correction. g , Western blotting of TASOR in primary MDMs treated for 24 hours with VLPs containing HIV-1 Vpr or control VLPs. h , Imaging flow cytometry of TASOR/Chromatin Similarity Score (co-localisation) in DAPI stained MDMs treated for 24 hours with VLPs. Mann-Whitney t-test. i , Representative images (60X magnification) of VLP treated MDMs from g and h which were treated Vpr-containing or empty VLPs. j , Fold increase in infectivity (p24 production over 48 hours) for HIV-1 89.6 WT and HIV-1 89.6 Δ vpr infected MDMs previously treated with HIV-1 Vpr containing VLPs relative to empty control VLP treated MDMs. Error bars represent standard deviations from the means of replicates. Mann-Whitney t test.

Article Snippet: Alkaline phosphatase-conjugated mouse anti-HIV-1 p24 monoclonal antibody (Aalto Bio Reagents) diluted in TBS 20% sheep serum, 0.05% v/v Tween-20 was then added and incubated for 1 hour at room temperature.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, shRNA, Control, Imaging, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: HIV-1 Vpr counteracts TASOR restriction to promote infection prior to integration

doi: 10.64898/2026.03.06.710055

Figure Lengend Snippet: HIV-1 Vpr overcomes early TASOR mediated restriction to reverse transcription. a , Western blotting of TASOR in HeLa-CD4 cells over time (0-24 hours) after challenge with HIV-1 89.6 WT or HIV-1 89.6 Δ vpr . b , Western blotting of MUS81 protein levels in THP-1 cells over the first 4 hours post challenge with HIV-1 89.6 WT . c , Western blotting of REAF and HLTF protein levels in THP-1 cells over the first 3 hours post challenge with HIV-1 89.6 WT (full blot +/- exogenous dN is in Figure S3). d , qPCR HIV-1 RT products – strong stop (left) and late (right) – 0–30 hr post infection (p.i.). Cells were treated with siRNA targeting TASOR (siTASOR) or a control gene Cyclophilin B (siCB). HIV-1 DNA copies measured by qPCR are normalised to genomic GAPDH and presented per 10 6 cells. Error bars represent standard deviations of means of duplicates. e , Western blotting of TASOR in PMA differentiated THP-1 cells over time (0-48 hours) after challenge with HIV-1 89.6 WT or HIV-1 89.6 R80A . p.i., post infection.

Article Snippet: Alkaline phosphatase-conjugated mouse anti-HIV-1 p24 monoclonal antibody (Aalto Bio Reagents) diluted in TBS 20% sheep serum, 0.05% v/v Tween-20 was then added and incubated for 1 hour at room temperature.

Techniques: Reverse Transcription, Western Blot, Infection, Control

Journal: bioRxiv

Article Title: HIV-1 Vpr counteracts TASOR restriction to promote infection prior to integration

doi: 10.64898/2026.03.06.710055

Figure Lengend Snippet: TASOR depletion causes cell cycle arrest and arrested cells are more susceptible to infection. a , Cell cycle analysis of DAPI stained HeLa (left) and HeLa-ΔTASOR (right) by imaging flow cytometry. b , Imaging flow cytometry of phosphorylated histone H3 (pH3 Ser28) in HeLa-shSCR, HeLa-shTASOR and HeLa-shREAF. Error bars represent the standard deviations of means of replicates. Kruskal-Wallis multiple comparison and ANOVA with Dunn’s correction. c , Proportions of untreated HeLa-CD4 (Ø) and thymidine/nocodazole (T/N) treated HeLa-CD4 cells in different phases of the cell cycle (G0/1, S/ G2, M) determined by flow cytometry of cells with DAPI staining for DNA quantification and staining of phosphorylated histone H3 Ser28 to identify cells in the mitotic phase (M). Western blotting of phosphorylated histone H3 Ser10/Thr11 in the same cells. d , Infectivity (FFU/ml) of HIV-1 89.6 WT in untreated HeLa-CD4 (Ø) and thymidine/nocodazole (mitotic cell enriched) HeLa-CD4 cells at three different virus inputs (dilution factor indicated). Fold changes in susceptibility to infection are shown. Error bars represent the standard deviations from the means of technical replicates. Unpaired t-tests.

Article Snippet: Alkaline phosphatase-conjugated mouse anti-HIV-1 p24 monoclonal antibody (Aalto Bio Reagents) diluted in TBS 20% sheep serum, 0.05% v/v Tween-20 was then added and incubated for 1 hour at room temperature.

Techniques: Infection, Cell Cycle Assay, Staining, Imaging, Flow Cytometry, Comparison, Western Blot, Virus

Journal: PLOS Pathogens

Article Title: The impact of clade B lineage 5 MERS coronaviruses spike mutations from 2015 to 2023 on virus entry and replication competence

doi: 10.1371/journal.ppat.1013336

Figure Lengend Snippet: (A-D) Cell entry of spike protein expressed lentivector pseudoviruses in Vero and Calu3 cells were measured by relative luciferase units (RLU). Spike expression plasmids encoding (A) clade B lineage 5 viruses from 2015 to 2018, (B) sequences from 2019, (C) individual single mutation from 2019 viruses, and (D) sequences from 2023, were co-transfected with third-generation lentivirus expression plasmids in 293T cells. In (A) - (D) , a normalized dose of 7-8 log 10 p24 RNA copies per 100ul of pseudovirus supernatant was added to target cells. Dotted horizontal line showed the RLU from “bald” non-spike expressed pseudovirus controls. Assays were performed with five replicates using two independent batch of pseudoviruses. (E) Western blot analysis of pseudovirus virions with different spike proteins. Spike proteins was stained by an anti-S2 antibody. The upper band corresponds to a full-length spike, whereas the lower band corresponds to a cleaved S2 protein. Virion quantity was measured by the levels of lentiviral p24 antigen. The data are from n = 4 independent replicate experiments and the blot shown is a representative example. The data are presented as the average ± SD. Statistically significant differences between 2015/GD01 and mutant spikes pseudoviruses were determined by two-sided Student’s t tests (* p < 0.05, ** p < 0.01, *** p < 0.001). Color legend: 2015/GD01 (blue), 2018 spikes (orange), 2019 spikes (brown) and 2023 spikes (magenta).

Article Snippet: Spike proteins were stained by primary anti-MERS-CoV S2 antibody (40070-T62, Sino Biological), followed by anti-Rabbit IgG secondary (#7074, Cell Signaling). p24 proteins were stained by primary anti-HIV-1 p24 antibody (sc-65918, Santa Cruz) and subsequently anti-mouse IgG secondary (#7076, Cell Signaling).

Techniques: Luciferase, Expressing, Mutagenesis, Transfection, Western Blot, Staining

Journal: PLOS Pathogens

Article Title: The impact of clade B lineage 5 MERS coronaviruses spike mutations from 2015 to 2023 on virus entry and replication competence

doi: 10.1371/journal.ppat.1013336

Figure Lengend Snippet: (A) 293T and stable expressing hDPP4 293T cells were infected with different spike expressing pseudoviruses at 37 o C. An equal normalized infection dose of 7-8 log 10 p24 RNA copies per 100μl was used for both cells and RLUs were measured at 48 hpi. RLU differences between 2015/GD01 and other spikes were determined by two-sided Student’s t tests (* p < 0.05, ** p < 0.01, *** p < 0.001). (B) FACS analysis of binding of recombinant S1-Fc proteins (at concentrations of 15 and 0.1 μg/ml) to Calu3 cells at 4 o C. Protein eluate from non-transfected control (NTC) (grey shading) served as the background signal.

Article Snippet: Spike proteins were stained by primary anti-MERS-CoV S2 antibody (40070-T62, Sino Biological), followed by anti-Rabbit IgG secondary (#7074, Cell Signaling). p24 proteins were stained by primary anti-HIV-1 p24 antibody (sc-65918, Santa Cruz) and subsequently anti-mouse IgG secondary (#7076, Cell Signaling).

Techniques: Expressing, Infection, Binding Assay, Recombinant, Transfection, Control