Dykddddk tag 9a3 mouse mab

The cells were washed three times with PBS and were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 20 min, blocked in PBST containing 5% BSA for 1 h at 37°C, and then incubated with primary antibody at 37°C for 1 h. Then, the cells were incubated with a second antibody at 37°C for 1 h. Actin filaments were stained with TRITC-phalloidin (2 μg/mL) for 30 min at 37°C. Cell nuclei were stained with DAPI (1 μg/mL) for 10 min. The cells were visualized using a confocal laser scanning microscope (LEICA TCS SP8, Germany).
HA-Tag (C29F4) rabbit MAb (Cell signaling technology. catalog number 3724) was used at a 1:200 dilution. DYKDDDDK Tag (9A3) mouse MAb (Cell Signaling Technology. catalog number 8146) was used at a 1:200 dilution. MYC-Tag antibody (Proteintech. catalog number 16286-1-AP) was used at a 1:200 dilution. NDV NP monoclonal antibody (gifted by Chan Ding) was used at a 1:100 dilution. Beta catenin polyclonal antibody (Proteintech. catalog number 51067-2-AP) was used at a 1:100 dilution. Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (Yeasen catalog number 33112ES60) was used at a 1:200 dilution. Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H+L) (Yeasen catalog number 33212ES60) was used at a 1:200 dilution. Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L) (Yeasen catalog number 33206ES60) was used at a 1:200 dilution.

KOD-Xtreme hot-start DNA polymerase (Merck Millipore), tiHybrid and Hybrid DNA Polymerases (EURx), SsoFast EvaGreen Supermix (BIO RAD), DreamTaq Green PCR Master Mix and Turbofect Transfection Reagent (ThermoFisher Scientific), Restriction endonuclease DpnI, EcoRI, and Gibson Assembly Master Mix (NEB), the plasmid used for substitution (GLuc-TEV-CBD) was designed in our laboratory, pmR-Cherry (Clontech), Coelenterazine (Selleckchem), human embryonic kidney-293T (HEK293T) cells (ATCC), Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium (Gibco), fetal bovine serum (FBS; PromoCell), ampicillin (Polfa Tarchomin), penicillin and streptomycin (Sigma Aldrich), DYKDDDDK Tag (9A3) Mouse mAb (Cell Signaling), Donkey Anti-Mouse IgG H&L Alexa Fluor 555 (Invitrogen).

Liver cancer cell lines (SMMC-7721, Huh7, MHCC-97 H, and HCC-LM3), normal hepatic cell line (LO2), and 293T cells were preserved in our laboratory and cultivated in DMEM (Hyclone) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were grown in an incubator of 5% CO₂ at 37 ℃. The pCMV3-Flag-CLEC1B plasmids were designed and confirmed by the Sino Biological (Beijing, China). The DYKDDDDK Tag (9A3) Mouse mAb (cat# 5750s, Cell Signaling Technology, China), CLEC1B Rabbit pAb (cat#A9971, ABclonal, China), and β-Actin rabbit mAb (cat# AC026, ABclonal, China) were applied for western blot analysis, and the dilution rate of the antibodies were 1:2,000, 1:1,000, and 1:5,000, respectively. Image J software was used for the quantification of western blot bands.