After treatment, BAECs were lysed in lysis buffer (20 mmol/l Tris–HCl pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l sodium orthovanadate, and 1% Triton X-100, supplemented with protease inhibitor cocktail) for 20 min on ice and then supernatant separated by centrifugation at 12,000 rpm for 10 min at 4°C. Following protein concentration determination by DC protein assay (#5000112, Bio-Rad, Hercules, CA, United States), 25–40 μg of protein were separated in 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by standard Western blotting protocol by probing with antibodies for ACE2 (1:1,000, #ab15348, Abcam, Waltham, MA, United States), TMPRSS2 (1:1,000, #ab92323, Abcam, Waltham, MA, United States), NOX2 (1:250, #611414, BD Biosciences, San Jose, CA, United States), MCP-1 (1:500, #ab9669, Abcam, Waltham, MA, United States), and β-actin (1:1,000, #A2066, MilliporeSigma, St. Louis, MO, United States) as we previously published (23 (link)). The protein bands were visualized by enhanced chemiluminescent methods, and band densities quantified using National Institutes of Health (NIH) Image J program.
Ab92323
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab92323 is a monoclonal antibody that targets the human protein Actin beta. This antibody is suitable for use in various immunoassays, including Western blotting and immunohistochemistry, to detect and quantify Actin beta expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab15348, by Abcam (9 mentions)
Ab108252, by Abcam (8 mentions)
Ab8245, by Abcam (3 mentions)
Ab81321, by Abcam (3 mentions)
Af933, by R&D Systems (3 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Ab172730, by Abcam (2 mentions)
Vector blue ap substrate kit 3, by Vector Laboratories (2 mentions)
Nuclear fast red, by Vector Laboratories (2 mentions)
H 5501, by Vector Laboratories (2 mentions)
Vectastain abc ap kit, by Vector Laboratories (2 mentions)
Anti g3pdh human polyclonal antibody, by Bio-Techne (2 mentions)
Biosciences, by LI COR (2 mentions)
Ab8226, by Abcam (2 mentions)
Bx46 microscope, by Olympus (2 mentions)
Dp27 camera, by Olympus (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Triton x 100 sample buffer, by Merck Group (2 mentions)
Ab49900, by Abcam (2 mentions)
Ab183495, by Abcam (2 mentions)
35 protocols using ab92323
1
Protein Expression Analysis in Endothelial Cells
2
Immunohistochemical Analysis of ACE2 and TMPRSS2 Expression
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Formalin-fixed paraffin-embedded tissue sections 5 μm thick were incubated in a 60° oven for 1 h and hydrated through a series steps of xylene and ethanol baths to water. Deparaffinized sections were boiled in antigen retrieval solution (100X citrate buffer, pH 6.0, Abcam, Cambridge, MA) for 10 min and cooled down for 20 min. Slides were then washed with hot tap water for 1 min and PBS twice for 3 min each time. After blocking in 5% normal goat serum diluted in PBS, the sections were incubated with anti-ACE2 antibody (ab108252, 1: 100, Abcam, Cambridge, MA), anti-TMPRSS2 antibody (ab92323, 1: 1000, Abcam) and the isotype- and concentration-matched normal IgG control (ab172730, Abcam) overnight at 4 °C. After 3 washes with PBST (PBS plus 0.1% Tween-20), the sections were incubated with the secondary biotinylated goat anti-rabbit IgG (1:2000) for 1 h at room temperature. The sections were then detected by alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA) and a blue reaction product was produced by incubating sections with alkaline substrate (Vector blue AP substrate kit III, Vector laboratories) for 20–40 min. The sections were also counterstained with nuclear fast red (Vector laboratories) and mounted with mounting medium (H-5501, Vector laboratories), and examined under a light microscope.
3
Western Blot Analysis of ACE, ACE2, and TMPRSS2
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Total cellular protein was extracted by dissolving cells in RIPA buffer. Protein concentration was measured using the Pierce BCA protein assay kit (23225, Thermo Fisher). Samples with normalized total protein concentrations were denatured at 95○C for 5 min and separated by SDS-PAGE, and then transferred to a PVDF membrane and blocked in 5 % skimmed milk in PBST for 1 h. Rabbit anti-ACE (1:1000, PA5–83080, Thermo Fisher), rabbit anti-ACE2 (1:1000, ab108252, Abcam), rabbit anti-TMPRSS2 (1:1000, ab92323, Abcam), mouse anti-β-actin (1:5000, Sigma) or mouse anti-α-tubulin (1:5000, Santa Cruz) in blocking buffer were added and incubated overnight at 4 oC. After washing with PBST, the membranes were incubated with anti-rabbit or anti-mouse secondary antibody conjugated with horse-radish peroxidase (1:5000, GE Healthcare) for 1 hr and washed. Membranes were developed using a chemiluminescence kit (Advansta, Griffin Biotech). The normalization of protein loading was confirmed with β-actin or α-tubulin expression.
4
Antibody-based Visualization of SARS-CoV-2 Targets
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The following antibodies were used: mouse anti‐ACE2 (Santa Cruz, sc‐390851, IF1:200), goat anti‐ACE2 (R&D systems, AF933, WB 1:1000), mouse anti‐dsRNA (Millipore, MABE1134, IF1:100), rabbit anti‐SARS‐CoV‐2 nucleocapsid protein/NP (Sino Biological, 40143‐R040, WB1:1000 IF1:200), rabbit anti‐TMPRSS2 (Abcam, ab92323, WB1:1000), rabbit anti‐cleaved caspase 3 (Cell Signaling, 9661 IF1:30 after pre‐labelling), rabbit anti‐GAPDH (Cell Signaling, 2118, WB1:2000), mouse anti‐ICAM‐1 (R&D systems, BBA3, IF1:200), mouse anti‐ICAM‐1 (Santa Cruz Biotechnology, sc‐8439, WB1:1000), Phalloidin‐Alexa555 (Cytoskeleton, PHDH1‐A, IF1:500), Phalloidin‐Alexa670 (Cytoskeleton, PHDN1‐A, IF1:200). Pre‐labelling of rabbit anti‐cleaved caspase 3 with Alexa Fluor 555 was performed using Zenon Rabbit IgG labelling kit (ThermoFisher, Z‐25305) according to manufacturer protocol.
5
SARS-CoV-2 Protein Expression Profiling
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On days 0, 1, 3, and 5 postinfection “Arlo” cells were lysed for Western blot analysis using Triton‐X‐100 sample buffer, as described previously.[130 ] Proteins were separated by SDS‐PAGE. Specific antibodies against SARS‐CoV‐2 N (1:1000 dilution, SARS‐CoV‐2 Nucleocapsid Antibody, Rabbit monoclonal antibody (Mab), #40143‐R019, Sino Biological), ACE2 (1:500 dilution, Anti‐ACE2 antibody, #ab15348, Abcam), TMPRSS2 (1:1000 dilution, Recombinant Anti‐TMPRSS2 antibody [EPR3861], #ab92323, Abcam), and GAPDH (1:1000 dilution, Anti‐G3PDH Human Polyclonal Antibody, #2275‐PC‐100, Trevigen) allowed antigen detection. Protein bands were made visible by laser‐induced fluorescence using an infrared scanner for protein quantification (Odyssey, Li‐Cor Biosciences).
6
TMPRSS2 and Androgen Receptor Expression
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Calu-3 (5 × 105 cells) were seeded in 6-well plates and incubated for 24 h for adhesion. After treatments, cells were lysed in RIPA buffer. Protein samples were then denaturated at 95 °C and migrated on 10% acrylamide/bisacrylamide gel. After transfer on a PVDF membrane (ThermoFisher Scientific), blocking was performed in 5% nonfat dry milk for 1 h. Membranes were then incubated overnight at 4 °C, with primary antibodies directed against the following proteins: TMPRSS2 (ab109131, 1/1000, Abcam, Cambridge, UK), TMPRSS2 Nt (ab92323,1/1000, Abcam, Cambridge, UK), AR (sc-816, 1/1000, Santa-cruz, CA, USA), GAPDH (ab8245, Abcam, Cambridge, UK) and β-actin (ab8226, 1/1000, Abcam, Cambridge, UK). Membranes were washed in TBS-tween before incubation with the corresponding HRP-conjugated secondary antibody for 1 h. Immune complexes were detected by chemiluminescence detection with Pierce ECL Western blotting substrate (ThermoFischer Scientific, Waltham, MA, USA) using G:BOX systems (Syngene). The quantification was carried out with the software ImageJ.
7
Immunohistochemical Analysis of ACE2 and TMPRSS2
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Hematoxylin and eosin staining and IHC on formalin-fixed tissues were performed by the Pathology Core at City of Hope. Antibodies used in IHC included: AR (SP107, Sigma-Aldrich), ACE2 (ab108252, Abcam), and TMPRSS2 (ab92323, Abcam). Slides were first reviewed at ×10 magnification to identify areas of positive staining, followed by confirmation and quantification at ×20 magnification. Representative images were acquired using an Olympus BX46 microscope with a DP27 camera at magnifications of ×20 and ×40, with a scale bar of 200 and 100 μm, respectively.
8
Western Blot Analysis of ACE2, TMPRSS2, Kim-1, and NRP-1
9
TMPRSS2 and AR Protein Analysis
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Cells were lysed in RIPA buffer (150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4) containing freshly added protease inhibitors (Halt Protease Cocktail, Thermo Fisher, Waltham, MA, USA). Samples were incubated on ice for 10 min, sonicated 4 cycles of 30 s on/off (Biorupter, Diagenode, Denville, NJ, USA) and centrifuged (21,100 ×g, 10 min, 4 °C). The DC protein assay (BioRad, Hercules, CA, USA) was used to quantify protein concentrations. Then, 30 μg of protein was separated using SDS-PAGE and immunoblotting performed65 (link). Primary antibodies used were rabbit anti-TMPRSS2 (1:100, ab92323, Abcam, Cambridge, UK), mouse anti β-actin (1:8000, ab8226, Abcam), rabbit anti-AR (1:2000, ab74272, Abcam) and mouse α-tubulin (1:10,000, B-5-1-2, Sigma Aldrich). Secondary HRP-conjugated antibodies (Sigma Aldrich) were used at 1:2000. Proteins were visualised using Immobilon Forte HRP substrate (Merck Millipore, MA, USA) and a Fusion FX Chemi Imager (Vilber Lourmat, Collégien, France).
10
Immunohistochemical Analysis of Lung Tissues
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Lung samples from the above mouse studies were fixed in 4% formalin for 24 h, before transfer to 70% alcohol for storage before processing into wax. Sections were probed with anti-Tmprss2 antibody (ab92323, Abcam) and anti-AR (ab108341, Abcam) overnight at 4 °C at a 1:1000 dilution before detection with the Histostain-Plus IHC HRP Kit and DAB (Thermo Fisher).
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