Gtx117425

Cells were lysed in lysis buffer [1% NP-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA] supplemented with a cOmplete ULTRA protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Cell lysates were separated by SDS-PAGE and transferred onto Immobilon-P PVDF membranes (Merck, Burlington, MA, USA). For detection of HA-tagged TTSPs which were transiently expressed in 293T-ACE2 cells, blotted membranes were incubated in HRP-conjugated anti-HA tag antibody (H6533, Sigma-Aldrich) diluted with 5% skim milk in TBS-T buffer [25 mM Tris-HCl (pH 7.5), 137 mM NaCl, 2.7 mM KCl]. For detection of TTSP-expression in Vero E6 cells, blots were incubated with primary antibodies: anti-TMPRSS2 (ab92323, Abcam, Cambridge, UK) antibody diluted with 5% skim milk in TBS-T buffer and anti-TMPRSS11D (GTX117370, GeneTex, Irvine, CA, USA), anti-TMPRSS11E (PA5-48775, Invitrogen, Waltham, MA, USA) and anti-TMPRSS13 (GTX117425, GeneTex) antibodies in Signal Booster (Beacle, Kyoto, Japan). The blots were then incubated with HRP-conjugated secondary antibodies. The HRP-conjugated anti-β-actin antibody (PM053-7, MBL, Nagoya, Japan) was used as a loading control. Signals were developed using Immobilon Western Chemiluminescent HRP Substrate (Merck).

Cells were lysed in lysis buffer (1% NP-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA) supplemented with cOmplete ULTRA protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lysate samples were resolved by SDS-PAGE and transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (Merck, Burlington, MA). Blots were incubated with the following primary antibodies: anti-TMPRSS2 (ab92323; Abcam, Cambridge, UK) antibody diluted with 5% skim milk in TBST (25 mM Tris-HCl [pH 7.5], 137 mM NaCl, 2.7 mM KCl) and anti-TMPRSS11D (GTX117370; GeneTex, Irvine, CA), anti-TMPRSS11E (PA5-48775; Invitrogen, Thermo Fisher Scientific), and anti-TMPRSS13 (GTX117425; GeneTex) antibodies diluted with Signal Booster (Beacle, Kyoto, Japan). Horseradish peroxidase (HRP)-conjugated anti-β-actin antibody (PM053-7; MBL, Nagoya, Japan) was used as a loading control. Immune complexes were detected using HRP-conjugated secondary antibodies and Immobilon Western chemiluminescent HRP substrate (Merck).