Hrp conjugated anti mouse igg

Vero-E6 cells were transfected with Ca_v1.2 α_1c-Flag. At 24 h after transfection, cells were inoculated with HRB25 (M.O.I. = 10) for 1 h on ice, and unbound virions were removed and fixed in 4% paraformaldehyde for 15 min. Multiplex immunofluorescence with Tyramide Signal Amplification was performed by following the previously established protocol [65 (link)]. The primary antibodies used in this study were anti-Flag (Sigma), anti-ACE2 (Abcam) and anti-SARS-CoV-2 nucleocapsid protein (Sino Biological). The secondary antibodies were HRP-conjugated anti-rabbit IgG (Zsbio) and HRP-conjugated anti-mouse IgG (Zsbio). Images were acquired using a Zeiss LSM880 laser-scanning confocal microscope equipped with Airyscan. Cells were scanned 24 layers along the Z axis with a pixel dwell time of 1 microsecond. The resolution of the acquired images was 2048 × 2048.
To quantify the colocalization of SARS-CoV-2, ACE2, and Ca_v1.2 α_1c-Flag, data from single channels were processed using the “surface module” of Bitplane Imaris software (Bitplane AG, Zurich, Switzerland), and then merged the produce images to observe the colocalization of SARS-CoV-2, ACE2, and Ca_v1.2 α_1c-Flag. The 3D-rendered images were generated by using Imaris software.

Total proteins of cells were extracted using RIPA lysis buffer (Beyotime) with Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche). Protein samples were separated in sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride (PVDF) filter membranes (Millipore, USA) for immune-hybridization. After 1 h of blocking in PBST (phosphate buffer saline containing 0.05% Tween-20 and 5% non-fat milk powder), the membranes were incubated with one of the following primary antibodies with corresponding concentration: RNF8 (Santa Cruz Biotech, 1:500), YBX1 (Cell Signaling Technology, 1:1000), HA (Convance, 1:1000), Secondary antibodies were Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (ZB-2305, ZSGB-Bio, 1:4000) or anti-Rabbit IgG(Fc) (ZB-2301, ZSGB-Bio, 1:4000). Subsequently, band visualization was performed by electro-chemiluminescence (ECL) and detected by Digit imaging system (Thermo, Japan), the gray level of the bands was quantitated by ImageJ software.

Total proteins were extracted with RIPA lysis buffer (Beyotime) with Protease Inhibitor Cocktail (Roche) and Phosphatase Inhibitor Cocktail (Roche). Protein samples were separated in sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride (PVDF) filter membranes (Millipore, USA). After blocking in phosphate buffered saline (PBS) containing 0.05% Tween-20 and 5% non-fat milk powder, the membranes were incubated with the following primary antibodies: RNF8 (Santa Cruz Biotech, 1:500), EMT kit (Cell Signaling Technology, 1:2000), beta-actin (Santa Cruz Biotech, 1:4000), Secondary antibodies were Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (ZB-2305, ZSGB-Bio, 1:4000) or anti-Rabbit IgG(Fc) (ZB-2301, ZSGB-Bio, 1:4000). Subsequent visualization was detected by Digit imaging system (Thermo, Japan), the gray level of the bands was quantitated by ImageJ software.