ace2 primary antibody  (Bioss)

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Immunofluorescence and Western blot analysis of ACE2 in HEK-293 and HEK-293/ACE2 cell lines. (A,C) Immunofluorescence localization of ACE2 proteins (red fluorescence). (B,D) Immunostaining images with primary antibodies omitted (control). All images show cell nuclei stained with DAPI (blue fluorescence). The scale bar represents 20 µm. (E) Representative immunoblots for ACE2 (90 kDa) and Actin (42 kDa) as a loading control are shown for HEK-293 cells and the stable cell line overexpressing human ACE2 (HEK-293/ACE2). (F) The relative abundance of ACE2 protein levels is expressed as the ratio of ACE2 to Actin band intensities. Data are shown as mean ± SEM from three independent experiments.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Immunofluorescence, Western Blot, Fluorescence, Immunostaining, Control, Staining, Stable Transfection

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Effect of bromhexine on SARS-CoV-2 Omicron pseudovirus infectivity in HEK-293/ACE2 cells. (A,C,E) Representative fluorescence microscopy images of cells infected with Omicron pseudovirus treated with 1, 10, and 100 µM bromhexine, respectively. (B) Positive control (Omicron pseudovirus infection without bromhexine). (D) Negative control (cells without Omicron pseudovirus infection or bromhexine). (F) Quantitative analysis of Omicron pseudovirus infection in HEK-293/ACE2 cells, based on the percentage of GFP-positive cells after infection. GFP expression indicates successful pseudovirus entry. Data are presented as means ± SEM ( n = 4) from at least three different experiments. An asterisk (*) indicates statistically significant differences ( p < 0.05) compared to the positive control, determined by one-way ANOVA with post hoc Tukey HSD test.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Infection, Fluorescence, Microscopy, Positive Control, Negative Control, Expressing

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Luciferase activity and IC 50 determination on HEK-293/ACE2 cells. (A) Infectivity of Omicron pseudoviruses was assessed by measuring luciferase activity in relative luminescence units (RLUs) after infecting cells with the viruses. Data are shown as means ± SEM ( n = 4). An asterisk (*) indicates statistically significant differences ( p < 0.05) determined by one-way ANOVA with post hoc Tukey HSD test. (B) Dose-response curve used to determine the half-maximal inhibitory concentration (IC 50 ) of bromhexine in HEK-293/ACE2 cells infected with Omicron pseudovirus, with an IC 50 of 17.3 ± 0.9 µM. Results are presented as means ± SEM ( n = 4). Curves are fitted to a 4-parameter logistic model and generated using the average of fitted parameters from individual experiments.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Luciferase, Activity Assay, Infection, Concentration Assay, Generated

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Reduction in infectivity of SARS-CoV-2 pseudovirus variants in HEK-293/ACE2 cells treated with bromhexine. Infectivity of pseudoviruses representing Alpha, Beta, and Delta SARS-CoV-2 variants was measured by luciferase activity, expressed in relative luminescence units (RLUs), 48 h after treatment with 40 μM bromhexine or a vehicle (control). The cells were infected with pseudoviruses engineered to express each variant. Data are shown as means ± SEM ( n = 4). An asterisk (*) indicates statistically significant differences ( p < 0.05) compared to the control group, determined by two-way ANOVA followed by post hoc Tukey HSD test.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Infection, Luciferase, Activity Assay, Control, Variant Assay

Journal: PLOS One

Article Title: pET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli , validated by production of functional human ACE2

doi: 10.1371/journal.pone.0327341

Figure Lengend Snippet: Protein expression as detected in total soluble protein extracts by western blot with anti-ACE2 antibody (A and B) and Coomassie Brilliant Blue staining (C and D) . A and C, soluble protein extracts of bacteria transformed with pET28a plasmid expressing ACE2 with a 6x-His tag at the N-terminus; B and D, soluble protein extracts of bacteria transformed with pET28g plasmids expressing ACE2 with an 8 × His-Gb1 tag (Gb1) or an MBP tag (MBP) at the N-terminus. NI – Soluble protein extract from bacteria before protein expression induction; I4 – Soluble protein extract from bacteria four hours after protein expression induction; M – Molecular weight marker (MB09002, NZYTech).

Article Snippet: Detection of ACE2 was performed using an anti-ACE2 primary antibody (1:2,000 dilution, AAF933 from R&D Systems) and HRP-conjugated anti-goat secondary antibody (1:20,000 dilution, sc-2020 from Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Staining, Bacteria, Transformation Assay, Plasmid Preparation, Molecular Weight, Marker

Journal: PLOS One

Article Title: pET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli , validated by production of functional human ACE2

doi: 10.1371/journal.pone.0327341

Figure Lengend Snippet: × His-Gb1 tag . A) SDS-PAGE analysis of fractions from the reverse HisTrap purification step, performed after TEV protease cleavage to remove the 8 × His-GB1 tag and His-tagged TEV protease. The flowthrough (FT) contains the cleaved (untagged) ACE2, while the elution fraction contains the His-tagged TEV protease and the cleaved His-tag. Washes 1 and 2 (W1, W2) with PBS pH 7.4 ensured complete recovery of untagged ACE2. The figure also shows fractions (a) and (b) collected from size-exclusion chromatography. (B) Size-exclusion chromatography profile of untagged ACE2 purified using a HiLoad 16/600 Superdex™ 200 pg column, where peak (b) corresponds to the untagged ACE2 protein. M – molecular weight marker (MB09002, NZYTech).

Article Snippet: Detection of ACE2 was performed using an anti-ACE2 primary antibody (1:2,000 dilution, AAF933 from R&D Systems) and HRP-conjugated anti-goat secondary antibody (1:20,000 dilution, sc-2020 from Santa Cruz Biotechnology).

Techniques: SDS Page, Purification, Size-exclusion Chromatography, Molecular Weight, Marker

Journal: PLOS One

Article Title: pET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli , validated by production of functional human ACE2

doi: 10.1371/journal.pone.0327341

Figure Lengend Snippet: (A) Far-UV CD spectra of ACE2 with a characteristic α-helical profile, indicating a predominantly helical secondary structure. Measurements were performed at a protein concentration of 0.2 mg/mL in PBS, pH 7.4, using a 0.1 cm path length cuvette at 25°C. Baseline correction was applied using the buffer CD spectrum. (B) Rates of Mca-APK(Dnp) substrate cleavage by recombinant ACE2. The rates are given in fluorescence arbitrary units (a.u.) because the unquenched Mca substrate was not available to prepare a calibration curve. For comparison, the rates observed for the control reactions, in the absence of substrate or protein, were plotted. Differences between the rate determined for ACE2 and the controls are statistically significant.

Article Snippet: Detection of ACE2 was performed using an anti-ACE2 primary antibody (1:2,000 dilution, AAF933 from R&D Systems) and HRP-conjugated anti-goat secondary antibody (1:20,000 dilution, sc-2020 from Santa Cruz Biotechnology).

Techniques: Circular Dichroism, Protein Concentration, Recombinant, Fluorescence, Comparison, Control