human ace2 his avi  (Bioss)

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Immunodetection of ACE2. ( a ) Vero or A549 cells were incubated with the indicated anti-ACE2 antibodies prior to fixation. Right panels show the background of the secondary anti-rabbit IgG antibody. Insets show the cell contours. ( b ) A549 cells cultured for 5 days or 7 days after plating were stained with the indicated anti-ACE2 antibodies after fixation with 4% (w/v) PFA and permeabilization with 0.1% (v/v) Triton X-100 for 5 min. Nuclei were counterstained with DAPI. (p, rabbit polyclonal; ab, rabbit polyclonal antibody from Abcam). Bars, 20 μm. ( c ) Detection of ACE2 by western blot. Total cell lysates from the indicated cell types containing 30 μg of protein, or 100 ng of purified ACE2 protein (ACE2-His) were analyzed by SDS-PAGE followed by immunoblot with the indicated anti-ACE2 antibodies. Left panels show a long and a short exposure of the blot of cell lysates and recombinant protein, respectively. Middle panels show a short exposure in both cases. In the right panel, the total protein on blots was visualized by staining with Simply Blue. ( d ) Scheme depicting the sequence of ACE2, the location of the epitopes of the antibodies used and the region spanned by the recombinant protein. SP, signal peptide; TM, transmembrane domain.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Immunodetection, Incubation, Cell Culture, Staining, Western Blot, Purification, SDS Page, Recombinant, Sequencing

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of Spike constructs and ACE2 in several cell types employing different sequences for immunodetection. ( a ) Scheme of the incubation and washing steps performed for immunodetection. Incubations were carried out for 1 h in the cold. After each incubation coverslips were washed three times with 200 μl of cold PBS. At the end of the procedure, an additional washing step with water was performed before coverslips were allowed to dry and and mounted. ( b ) Representative images from the detection of Spike constructs and ACE2 in the indicated cell lines employing the different immunodetection sequences. The graph shows the colocalization between Spike S1-Fc and ACE2 fluorescent signals for every cell type and immunodetection sequence assayed. Colocalization is expressed as the proportion of Spike S1 colocalizing with ACE2 signal (Manders’ coefficient), measured applying the automatic Costes’ threshold (n ≥ 8 per condition). Results are shown as mean values ± SEM; ns, non-significant, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 by ANOVA with Tukey’s post-test. Bars, 20 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Construct, Immunodetection, Incubation, Sequencing

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of vimentin and ACE2 in A549 cells. ( a ) Live A549 cells were incubated simultaneously with anti-vimentin and anti-ACE2 antibodies, after which they were incubated with a combination of the corresponding secondary antibodies prior to fixation. An illustrative image is depicted showing points of colocalization at intercellular contacts at mid-cell height, highlighted in the colocalization mask. In addition, an overlay of the region of interest delimited by the dotted square with the bright field image is shown to illustrate the juxtanuclear position of one of the colocalization points (white arrow); the contour of the nucleus is highlighted in magenta. The far right panel shows the top section of the region of interest along with the orthogonal projections centered in the colocalization point marked with the arrow. Bars, 20 μm. ( b – d ) Cells were fixed and permeabilized as specified below before immunodetection. ( b ) A549 cells were fixed with 4% (w/v) PFA and permeabilized with 0.1% (v/v) Triton for 5 min for staining with monoclonal antibodies against acetylated tubulin (acTubulin, left panels) or ARL13B (right panels). Nuclei were stained with DAPI. ( c ) A549 cells fixed and permeabilized as in ( b ), were stained with anti-ACE2 and anti-acetylated tubulin antibodies. ( d ) A549 cells were fixed with 2% (w/v) PFA, permeabilized with 0.1% (v/v) Triton X-100 and stained with anti-vimentin antibodies (SP20 or C-end) and either anti-acetylated tubulin or anti-ARL13B antibodies, as indicated. Images in ( b – d ) are single sections. In each case, the regions of interest (dotted square) are enlarged at the right. Bars, 10 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Incubation, Immunodetection, Staining

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of Spike S1, ACE2 and vimentin at primary cilia. A549 cells were incubated with Spike S1-Fc at 37 °C for 90 min in complete medium. Non-permeabilized cells were stained with ( a ) anti-ACE2 and anti-vimentin 84-1 antibodies, ( b ) anti-ACE2 and anti-acetylated tubulin or ( c ) anti-vimentin SP20 and anti-ARL13B antibodies, followed by the corresponding secondary antibodies, and finally with anti-human IgG antibody to detect the Spike S1 Fc tag, before fixation. In ( a , b ) cells were fixed with 4% (w/v) PFA and in ( c ) with 2% (w/v) PFA. Images shown are single sections for each channel obtained by confocal microscopy, at middle height of the cells, and the corresponding overlays. Regions of interest (dotted squares) are enlarged in the lower panels for each condition. Colocalization masks for the signals of ( a ) vimentin and Spike, ( b ) ACE2 and Spike, and ( c ) vimentin and Spike, are shown at the right as white signals on black background; numbers in insets correspond to the Pearson’s coefficient and the percentage of colocalization for the regions shown. Colocalization analysis was performed with Leica software. Arrows in c point to structures showing colocalization. Bars, 10 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Incubation, Staining, Confocal Microscopy, Software