Anti flag

Antibodies used in this article include anti-TRAF4 (Proteintech: Wuhan, China, CAS: 66755-1-Ig), anti-TRAF4 (Abcam: NY, USA, CAS: ab245666), anti-Tubulin (Proteintech: Wuhan, China, CAS: 66031-1-Ig), anti-E-cadherin (Proteintech: Wuhan, China, CAS: 20874-1-AP), anti-MMP2 (Proteintech: Wuhan, China, CAS: 10373-2-AP), anti-β-catenin (Cell Signaling Technology: Danvers, MA, USA, CAS: 8480), anti-CCND1 (Proteintech: Wuhan, China, CAS: 60186-1-Ig), anti-GSK3β (Proteintech: Wuhan, China, CAS: 22104-1-AP), anti-p-GSK3β (Cell Signaling Technology: Danvers, MA, USA, CAS: 5558), anti-AKT1 (Proteintech: Wuhan, China, CAS: 60203-2-Ig), anti-p-AKT (Cell Signaling Technology: Danvers, MA, USA, CAS: 13038), anti-SETDB1(Cell Signaling Technology: Danvers, MA, USA, CAS: 2196), anti-Flag (Proteintech: Wuhan, China, CAS: 66008-3-Ig), anti-Flag (Proteintech: Wuhan, China, CAS: 80010-1-RR), anti-MYC-tag (Proteintech: Wuhan, China, CAS: 60003-2-Ig), anti-MYC-tag (Proteintech: Wuhan, China, CAS: 16286-1-AP), and anti-HA (Proteintech: Wuhan, China, CAS: 51064-2-AP).
Reagents used in this article including Cycloheximide (CHX) (Merck: Kenilworth, NJ, USA, CAS: 66-81-9) and proteasome inhibitor (MG-132) (MCE: Chongqing, China, CAS: 133407-82-6).

To determine whether Nsp14 co-localizes with chJAK1, the HD11 cells were planted on glass coverslips and grown overnight in 35 mm cell culture dishes. The cells were transfected with pcDNA3.1-Flag-Nsp14 and pCMV-Myc-chJAK1 for 24 h and washed three times with cold PBS. They were treated for 15 min at room temperature with 4% paraformaldehyde. Then, the cells were incubated with anti-Flag antibodies and rabbit polyclonal anti-Myc primary antibodies (Proteintech) (1:100) for 1 h.
To explore the effect of Nsp14 on nuclear translocation of chSTAT1, HD11 cells were transfected with pcDNA3.1-Flag-Nsp14 for 24 h, stimulated with chIFN-γ (100 ng/mL) for 30 min before being washed three times with cold PBS, and treated for 15 min at room temperature with 4% paraformaldehyde. The cells were then incubated with anti-Flag (Proteintech) and anti-chSTAT1 (Bioss) antibodies (1:100) for 1 h.
All the cells were washed with PBS and incubated with secondary antibodies (CoraLite488-conjugated donkey anti-mouse IgG (H + L) and CoraLite594-conjugated donkey anti-rabbit IgG (H + L) (1:200)) for 1 h. Then, the cells were dyed with DAPI (Beyotime) for 10 min and washed with cold PBS. The images were captured using a laser scanning confocal microscope (Zeiss, Jena, Germany).