Journal: PLoS ONE

Article Title: Dysregulation of microRNAs and renin-angiotensin system in high salt diet-induced cardiac dysfunction in uninephrectomized rats

doi: 10.1371/journal.pone.0180490

Figure Lengend Snippet: 60 μg of protein was loaded per each lane. To ensure the correct position of the protein of interest, a pre-stained protein marker (Invitrogen Novex ® Sharp Pre-stained Protein Standard, Cat # LC5800, Thermo Fisher Scientific) was run along with the samples in the gel. The results were expressed as -fold change over control rats. A) Immunoblots of heart. B) Quantification of western blots of SERCA2 (normalized with actin), p-AKT1 (Ser 473) (normalized with AKT) p-PTEN (Ser 380) (normalized with actin) p-AMPK (normalized with AMPK). C) Immunoblot of renal ACE2 (normalized with actin). D), E) Relative levels of miR-25, miR-155, miR-451 and miR-99b in heart normalized with sn-RNU5G; F) Relative levels of miR-25, miR-451 and miR-99b in plasma normalized with miR-30e. N = 3–5.*P<0.05, **P<0.01, ***P<0.001 vs. Ctrl; S P<0.05, SS P<0.01, SSS P<0.001 vs. HSD; U P<0.05, UU P<0.01, UUU P<0.001 vs. UNX.

Article Snippet: These were then electrotransferred to nitrocellulose membranes and were incubated with below mentioned antibodies—ACE2 (rabbit pAb, Cat# sc-20998), p-AMPK (rabbit pAb, sc-33524), AMPK (rabbit pAb, sc-25792), p-AKT (Rabbit pAb, SAB4300042), AKT (rabbit pAb, SAB4500802), actin (goat pAb, sc-1616, SantaCruz Biotechnology, Inc., CA, USA), SERCA = ATP2A2 (rabbit pAb, Cat# HPA062605-100UL, Sigma-Aldrich, MO, USA).

Techniques: Staining, Marker, Control, Western Blot, Clinical Proteomics

Journal: Pathogens

Article Title: InfectionCMA: A Cell MicroArray Approach for Efficient Biomarker Screening in In Vitro Infection Assays

doi: 10.3390/pathogens11030313

Figure Lengend Snippet: ICC results for ACE2 and NRP1 proteins in SARS-CoV-2-infected Vero cells, at various time points (4 h, 24 h, 48 h and 72 h), for mock (non-infected), MOI 0.001 and MOI 0.01. ( A ) Microscopic images with a 50 µm scale bar. The last column represents zoomed-in images of the sections highlighted within a red square in the respective rows. Both viral proteins were located in the cytoplasm of the infected cells. ACE2 was localized in the membrane, and NRP1 in the membrane and in the cytoplasm. ( B ) Heat map for the percentage of cells stained (0–1%, >1–10%, >10–25%, >25–50%, >50–75%, and >75%).

Article Snippet: The InfectionCMAs were immunostained with the following primary antibodies: SARS-CoV-2 spike [1A9] (mouse; GTX632604, Genetex, Irvine, CA, USA), SARS-CoV-2 nucleocapsid [6H3] (mouse; GTX632269, Genetex, Irvine, CA, USA), ACE2 (mouse; MA5-31395, Thermo Scientific, Waltham, MA, USA), Neuropilin 1 [EPR3113] (rabbit; ab183495, Abcam, Cambridge, UK), Ki-67 (rabbit; MA5–14520, Thermo Scientific, Waltham, MA, USA) and BCL2 (rabbit; AB196495; Abcam, Cambridge, UK).

Techniques: Infection, Membrane, Staining

Journal: Pathogens

Article Title: InfectionCMA: A Cell MicroArray Approach for Efficient Biomarker Screening in In Vitro Infection Assays

doi: 10.3390/pathogens11030313

Figure Lengend Snippet: Characterization of ACE2 expression in the human (Caco-2 and Huh-7) and Vero cell lines, at various time points after infection with MOI 1, by ICC (( A ) microscopic images with 100 µm scale bar, including zooms of the red inserts; ( B ) heat map), Western blot ( C ), and qRT-PCR ( D ). In the graphs, data represent means ± SD of three independent experiments, and significant Student’s t -test p -values are indicated (** < 0.01). The ACE2 was placed in the membrane of all the infected cell lines.

Article Snippet: The InfectionCMAs were immunostained with the following primary antibodies: SARS-CoV-2 spike [1A9] (mouse; GTX632604, Genetex, Irvine, CA, USA), SARS-CoV-2 nucleocapsid [6H3] (mouse; GTX632269, Genetex, Irvine, CA, USA), ACE2 (mouse; MA5-31395, Thermo Scientific, Waltham, MA, USA), Neuropilin 1 [EPR3113] (rabbit; ab183495, Abcam, Cambridge, UK), Ki-67 (rabbit; MA5–14520, Thermo Scientific, Waltham, MA, USA) and BCL2 (rabbit; AB196495; Abcam, Cambridge, UK).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR, Membrane

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 1 | SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Binding Assay, Clone Assay, Marker, Magnetic Beads

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 5 | SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B), except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase If, a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Western Blot, Activity Assay, Magnetic Beads, Labeling, Positive Control

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 6 | Expression of hRNase A-ACE2NTD150 in T7 Express LysY/lacIq (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot