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5 protocols using anti tmprss2

1

Western Blot Analysis of Cellular Proteins

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The treated cellular protein extracts were prepared as described previously (Lin et al., 2017 (link); Lee et al., 2018 (link)). In brief, equal amounts of protein were separated on an 8–10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried milk for 30 min and then incubated with primary antibodies for 6–12 h at room temperature. The following primary antibodies were used: anti-ACE2 (1:1,000; Cell Signaling), anti-STAT3 (1:1,000; Cell Signaling), anti-TMPRSS2 (1:1,000; Abcam), anti-β-actin (1:10,000; Santa Cruz), and anti-GAPDH (1:10,000; Santa Cruz) antibodies. All the primary and secondary antibodies were diluted with 1% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 detergent. The membranes were washed using 0.1% Tris-buffered saline with Tween-20 and incubated in horseradish peroxidase–conjugated secondary anti-mouse or anti-rabbit antibodies (Santa Cruz, ratio: 1:5,000) for 1 h at room temperature. The membranes were washed for 1 h at room temperature. Chemiluminescent protein signals were detected by applying the SuperSignal West Pico PLUS chemiluminescent substrate (Pierce, catalog number: 34087).
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2

Corneal ACE2 and TMPRSS2 Protein Analysis

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Corneal epithelial cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail. The protein lysate was mixed with loading buffer, electrophoresed via SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked and then incubated with primary antibodies (anti–ACE2 [15348; Abcam, Cambridge, MA, USA], anti-TMPRSS2 [Abcam, 92323] and an anti-GAPDH antibody [CB1001; Merck Millipore, Burlington, MA, USA]). GAPDH was used as an internal control. The intensity of the immunoreactive bands was analyzed using ImageJ.
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3

Antibody Detection for SARS-CoV-2 Targets

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Anti-ACE2 were purchased from Abcam (ab108209), Anti-CTSL were purchased from R&D(AF952-SP), Anti-β-actin were purchased from sigma(A5316), Anti-TMPRSS2 were purchased from Abcam (ab109131).
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4

Multimodal IHC for COVID-19 Markers

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Formalin (4%) fixed duodenum and colon tissue samples were embedded in paraffin, and a 4 μm section of each was obtained on a glass slide. These sections were deparaffinized and incubated with anti-HIV p24 (clone: Kal-1, Dako), anti–SARS-CoV-2 nucleoprotein (clone: 40143-T-62, Sino Biological) anti-ACE2 (clone: ab15348; Abcam), anti-TMPRSS2 (clone: ab109131; Abcam), and anti-EpCAM (clone: ab71916; Abcam) followed by a secondary antibody incubation using the Opal 4-color manual IHC (PerkinElmer) as instructed by the manufacturer. For Opal fluorophores (PerkinElmer), FITC (product number FP1487001KT; PerkinElmer) was used for EpCAM and p24, Texas red (product number FP1488001KT; PerkinElmer) for ACE2, and Cy5 (product number FP1497001KT; PerkinElmer) for TMPRSS2 and SARS-CoV-2 nucleoprotein signal generation. DAPI was used as the nuclear counterstain. The sections were mounted with the Fluorescence Mounting Medium (catalog number S302380-2; Agilent Technologies) and cover-slipped, and the edges were sealed with nail polish. The slides were stored at 2°C–8°C until images were acquired.
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5

SARS-CoV-2 Host Factor Protein Detection

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Cells were lysed in RIPA buffer with protease inhibitor cocktail (Pierce) and centrifuged 14,000 rpm for 15 min at 4 °C. Cell lysates were mixed with NuPage LDS sample buffer supplemented with a reducing agent (Life Technologies) and boiled for 10 min. Next, 7–8 μg of protein was resolved on 4–12% Bis-Tris gels (Life Technologies) and transferred to a PVDF membrane using X-cell surelock mini transfer appartus (Life Technologies) at 22V for 1 h. Membranes were blocked with 5% skimmed milk (BioShop) in 1X TBST prior to antibody incubation. Subsequently, proteins were detected using anti-ACE2 (CST 92485), anti-TMPRSS2 (AbCam ab109131), anti-CTSL (R&D AF952), α-tubulin (MilliporeSigma sc-53646), anti-HSP90 (SantaCruz, SC-13119) antibodies with an appropriate HRP-conjugated secondary antibody, anti-mouse IgG (Cell Signalling Technologies 7076, RRID:AB_330924), anti-rabbit IgG (Cell Signalling Technologies 7074, RRID:AB_2099233) or anti-goat IgG (R&D HAF017). Proteins were visualized on iBright Imaging Systems (ThermoFisher Scientific) using Pierce SuperSignal West Pico ECL reagents (ThermoFisher Scientific).
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