Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Schematic of the screening for candidate membrane proteins involved in SARS-CoV-2 entry. Schematic illustration of the labeling procedure according to EMARS. After EMARS reaction, the fluorescein-labeled proteins were purified and then analyzed using mass spectrometry.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Purification, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: SARS-CoV-2 spike protein-based EMARS probes. ( A ) ACE2 expression in Caco-2 and A549 cells. Western blot analysis of Caco-2 and A549 cell lysates; 10 μg protein samples were subjected to SDS-PAGE (on 10% gels) and stained with anti-ACE2 antibody. Arrows indicate bands of the ACE2 protein. ( B ) Immunocytochemical staining of ACE2 in Caco-2 and A549 cells. Staining with the anti-ACE2 antibody (ACE2+2 nd 568) was performed as described in Experimental procedure . Negative control samples (2 nd 568) were also prepared simultaneously. White bar: 100 μm. ( C ) Immunocytochemical staining of SARS-CoV-2 spike proteins in Caco-2 and A549 cells. Staining of monovalent Alexa Fluor 488-labeled spike proteins (spike-488) and the two-step staining (spike protein followed by Alexa Fluor 488 secondary antibody; spike+2 nd 488) were performed with DIC images. Negative control samples (cAb-488 or 2 nd 488) were also prepared simultaneously. White bar: 100 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Expressing, Western Blot, SDS Page, Staining, Negative Control, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Proximity labeling near the cell membrane-bound SARS-CoV-2 spike protein. ( A, B ) Fluorescein-labeled proximal proteins around cell membrane-bound SARS-CoV-2 spike proteins. The EMARS reaction described in the “Experimental procedure” was performed in Caco-2 ( A ) and A549 ( B ) cells using a spike protein ( Spike (RBD) ) and HRP-conjugated anti-mouse IgG ( mouse HRP ). The EMARS products were subsequently subjected to Western blot analysis to detect fluorescein-labeled proteins as candidate proximal proteins. In Caco-2 cells, HRP-conjugated Cholera Toxin B Subunit B ( CTxB-HRP ) was used for EMARS reaction as the positive control for membrane protein labeling. For loading controls, the PVDF membrane was stained with Coomassie Brilliant Blue after western blot analysis (right column)

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Western Blot, Positive Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Co-localization of the identified proteins with cell membrane-bound SARS-CoV-2 spike proteins. Representative images of co-localization with SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells were co-stained for SARS-CoV-2 spike protein (green) and the antibodies recognizing ACE2, CD133, Cadherin 17, DPP4, and VAPA (Red). The resulting specimens were subsequently stained with appropriate secondary antibodies and DAPI (Blue), then observed using confocal microscopy (20× objective). Co-localization is indicated in yellow in the “Merge” images. White bar: 10 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Candidate proteins located near SARS-CoV-2 spike proteins. ( A to D ) Morphological observation of SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells observed using electron microscopy. Cultured Caco-2 cells were fixed and co-stained with the SARS-CoV-2 spike protein (indicated as 20 nm particles), and candidate molecules identified. CD133 ( A ), DPP4 ( B ), CDH17 ( C ), and VAPA ( D ) are indicated as 10 nm particles. Red arrows indicate the locations of SARS-CoV-2 spike proteins. Yellow arrow heads indicate the location of each candidate protein. Scale bar; 200 or 500 nm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Electron Microscopy, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: In vitro infection assay of SARS-CoV-2 pseudovirus. ( A ) Expression of ACE2 and candidate membrane proteins in transfectant HEK293 cells. Western blot analysis of transfectant cell lysates; Each cell lysates were subjected to SDS-PAGE (on 6 to 10% gels) and stained with antibodies recognizing ACE2 or candidate membrane proteins. Arrows indicate bands of the target proteins. The CBB staining image indicates load control. Asterisks indicate predicted nonspecific bands. ( B ) Schematic illustration of the assay procedure using HEK293T transfectant host cells. ( C ) Representative images of GFP-positive P-ACE2 cells after pSARS-CoV-2 infection. ACE2-expressing HEK293T cells were treated (pSARS-CoV-2 (+)) or not treated (pSARS-CoV-2 (-)) with pSARS-CoV-2, followed by fluorescein microscopic observation. Two independent experiments were carried out. White bar: 100 μm. ( D-F ) Flow cytometric analysis of pSARS-CoV-2-infected cells. P-ACE2 cells ( D ), candidate protein-single expressing cells ( E ), and candidate protein-coexpressing P-ACE2 cells ( F ) were analyzed using BD FACS Canto II. GFP-positive cells were defined as the infected cells with a GFP fluorescence intensity of 10 3 or higher (P3 area). Two ( E ) or five ( D and F ) independent replications were carried out in each experiment. ( G ) Increase in pSARS-CoV-2 infection in candidate protein-coexpressing P-ACE2 cells. The number of GFP-positive cells in each cell was quantified using flow cytometry. The number of infected cells (GFP-positive) in P-ACE2–CD133, –CDH17, and –VAPA was significantly higher than that in P-ACE2 cells ( P < 0.05 or P < 0.005; Dunnett's test), but not in P-ACE2-GPC3 (N.D.) as the negative control.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: In Vitro, Infection, Expressing, Transfection, Western Blot, SDS Page, Staining, Fluorescence, Flow Cytometry, Negative Control

Journal: Journal of Ovarian Research

Article Title: Human amniotic mesenchymal stem cell-derived extracellular vesicles improve oocyte quality and embryonic development by increasing antioxidant capacity in aged mice

doi: 10.1186/s13048-025-01913-x

Figure Lengend Snippet: Isolation and characterization of hAMSC-EVs. A Flow cytometric analysis of hAMSCs revealed that hAMSCs exhibited high expression of CD73, CD44, CD105, and CD90 (> 95%), and very low levels of CD11b, CD19, CD34, CD45, and HLA-DP/DQ/DR (< 2%). B AMSCs exhibited fibroblast morphology and adherent behavior. Scale bar:100 μm. C hAMSC-EVs were cup-shaped under transmission electron microscopy. D hAMSC-EVs had a diameter of 126.5 ± 6.35 nm, as determined by nanoparticle tracking analysis. E hAMSC-EVs positively expressed the EV-specific proteins CD9, TSG-101 and Calnexin according to immunoblotting analysis; hAMSC-EVs: extracellular vesicles derived from human amniotic mesenchymal stem cells

Article Snippet: The gels were transferred onto polyvinylidene difluoride membranes at 350 mA for 70 min, blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature (RT), and then incubated with primary antibodies against beta-actin (ab6276, Abcam), CD9 (sc-13118, Santa Cruz Biotechnology, Inc.), TSG-101 (ab30871, Abcam), Calnexin (ab22595), NRF2 (16396-1-AP, Proteintech), KEAP1 (60027-1-lg, Proteintech)and SOD2 (24127-1-AP, Proteintech) overnight at 4 °C.

Techniques: Isolation, Expressing, Transmission Assay, Electron Microscopy, Western Blot, Derivative Assay

Journal: Chinese medicine

Article Title: Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis.

doi: 10.1186/s13020-023-00876-9

Figure Lengend Snippet: Fig. 3 Autophagy is implicated in lipid droplet degradation under combined treatment of T0 and MGF. Representative digital images of electron microscopy reveal autophagic vacuoles accumulating in the cytoplasm of peritoneal macrophages (A) and RAW264.7 cells (B). Scale bar, 50 μm, 1 μm, 500 nm. Expression of p-mTOR, mTOR, p-AMPK, and AMPK in peritoneal macrophages (C) and RAW264.7 cells (D) was determined by western blot with total proteins extracted from cell samples. Expression of p62, LC3, Beclin1 and ATG5 in peritoneal macrophages (E) and RAW264.7 cells (F) was determined by western blot with total proteins extracted from cell samples

Article Snippet: Primary rabbit polyclonal antibodies against α-SMA (14395), CD36 (18836), Beclin1 (11306), ATG5 (10181), and ATG7 (10088) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA).

Techniques: Electron Microscopy, Expressing, Western Blot

Journal: Frontiers in Microbiology

Article Title: The role of SIRT1 in the process of Toxoplasma gondii infection of RAW 264.7 macrophages

doi: 10.3389/fmicb.2022.1017696

Figure Lengend Snippet: Murine ortholog of immunity-related GTPase family M (IRGM1) regulation by Sirtuin 1 (SIRT1) with or without interferon gamma (IFN-γ) or CD154 at different time points. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with IFN-γ (1 μg/ml), CD154 (200 ng/ml), SRT1720 (5 μM), EX527 (10 μM), IFN-γ (1 μg/ml) and SRT1720 (5 μM), or IFN-γ (1 μg/ml) and EX527 (10 μM) for 2, 18, or 24 h before the collection of the cell lysates. We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-IRGM1 antibody and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) The expression of IRGM1 in the Toxoplasma gondii infection group increased at 2 h but decreased at 18 and 24 h compared to that for the control group. Infected macrophages treated with IFN-γ displayed an upregulation of IRGM1 at 2, 18, and 24 h compared to that of the infection-only group. (B) Infected macrophages treated with CD154 showed no significant changes of IRGM1 compared to that for the infection-only group. (C) Infected macrophages treated with RH tachyzoites showed an upregulation of IRGM at 2 h and a downregulation at 18 or 24 h compared to that of the control group. Meanwhile, IRGM1 production was found to be decreased under SRT1720 stimulation and increased with the existence of EX527 at 2 h. However, both SRT1720 and EX527 failed to interfere with the IRGM1 level at 18 and 24 h. (D) When combining SIRT1 interference and IFN-γ stimulation, infected macrophages treated with IFN-γ and SRT720 showed a downregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. Meanwhile, infected macrophages treated with IFN-γ and EX527 showed an upregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. These results were obtained from three independent experiments. RH, T. gondii RH strain. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following reagents and equipments were used in the studies: SRT1720 (hydrochloride) (CAS No.: 1001645, MCE, USA), EX527 (CAS No.: 49843-98-3, MCE, USA), Rapamycin (CAS No.: 53123-88-9, MCE, USA), recombinant Interferon-γ (Cat. No.: I4777, Sigma-Aldrich, USA), recombinant mouse TRAP/CD40L protein (Active) (ab220551, Abcam, USA), anti-SIRT1 antibody [SirT1 (1F3) Mouse mAb #8469, Cell Signaling Technology, USA], anti-LC3 antibody [LC3A/B (D3U4C) XP Rabbit mAb #12741, Cell Signaling Technology, USA], anti-mTOR antibody [mTOR (7C10) Rabbit mAb #2983, Cell Signaling Technology, USA], anti-Phospho-mTOR antibody [Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb #5536, Cell Signaling Technology, USA], anti-IRGM1 antibody [IRGM (E6P7W) Rabbit mAb #71950, Cell Signaling Technology, USA], anti-beta-Actin mouse monoclonal antibody (Cat. No.: T0022, Affinity, China), LysoTrackerTM Red DND-99–Special Packaging (Cat. No.: L7528, Thermo Fisher, USA), ProLong Gold Antifade Mountant with DAPI (Cat. No.: P36935, Thermo Fisher, USA), Goat anti-Mouse Alexa Fluor 488 (Cat. No.: A-11017, Thermo Fisher, USA), microplate reader (Sunrise, TECAN, Switzerland), Confocal Microscope (LSM 710, Zeiss, Germany), and Transmission Electron Microscope (TEM) (HT7800, Hitachi, Japan).

Techniques: Infection, Incubation, Protein Concentration, Expressing, Control

Journal: Diagnostics

Article Title: Performance of Antigen Detection Tests for SARS-CoV-2: A Systematic Review and Meta-Analysis

doi: 10.3390/diagnostics12061388

Figure Lengend Snippet: Characteristics of the 235 studies included in the meta-analysis.

Article Snippet: Miyakawa et al. [ ] , Japan , N , nsp , LFIA/virus culture data , 1. SARS-CoV-2 Ag-RDT 2. Panbio COVID-19 Ag Rapid Test 3. SARS-CoV-2 Rapid Antigen Test 4. SD Biosensor Standard Q COVID-19 Ag 5. Espline SARS-CoV-2 , 1. YCU-FF 2. Abbott 3. Roche 4. SD Bio 5. Fujirebio , Up to 40 , Rapid , 108 , 45 , 63.

Techniques: Diagnostic Assay, Surround Optical-fiber Immunoassay, Fluorescence, Enzyme-linked Immunosorbent Assay, Raman Spectroscopy, Antigen Assay, Mass Spectrometry, Stripping Membranes