Anti becn1

The following primary antibodies were used in the study: rabbit polyclonal anti-LC3B (Cell Signaling Technology, 2775), rabbit polyclonal anti-SQSTM1/p62 (Sigma, SAB2104334), rabbit polyclonal anti-BECN1 (ABclonal, A7353), rabbit polyclonal anti-ATG5 (ABclonal, A0203), mouse monoclonal anti-GAPDH (Beyotime, AG019), rabbit polyclonal anti-caspase-8 (Beyotime, AC056), rabbit polyclonal anti-caspase-9 (Beyotime, AC062), rabbit polyclonal anti-Caspase-3 (ABclonal, A2156), rabbit polyclonal anti-PARP (Beyotime, AP102) and mouse monoclonal anti-CSFV E2 (WH303) (JBT, 9011). Mouse polyclonal anti-CSFV Npro was kindly provided by Dr. Xinglong Yu (Veterinary Department, Hunan Agricultural University, China). The secondary antibodies used for immunoblotting analysis were HRP-conjugated goat anti-mouse IgG (Bioworld, BS12478), HRP-conjugated goat anti-rabbit IgG (Bioworld, BS13278). The secondary antibodies used for immunofluorescence including Dylight 488 goat anti-mouse IgG (EarthOx, E032210), Dylight 488 goat anti-rabbit IgG (EarthOx, E032220), Dylight 549 goat anti-mouse IgG (EarthOx, E032310) and Dylight 549 goat anti-rabbit IgG (EarthOx, E032320).

Cells were harvested and lysed in lysis buffer for protein extraction. Protein concentrations were measured by BCA protein assay kit (Thermo Scientific). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies: rabbit anti-SIRT1 (Millipore), anti-BECN1 (Abclonal Technology), anti-LC3B (Cell Signaling Technology), anti-p16 (Abclonal Technology), anti-p21 (Abclonal Technology), anti-p53 (Abclonal Technology), and mouse anti-β-actin (Sigma) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP-donkey-anti-rabbit and HRP-donkey-anti-mouse) (Santa Cruz Biotechnology). Signal was detected by an ECL kit. The experiments were performed three times.

Zebrafish gene mylipb (ENSDARG00000055118) and its truncated mutants were PCR amplified from AB zebrafish cDNA using PCR. Amplified genes were subcloned into pCMV-Myc (Clontech), pCMV-HA (Clontech), or pCMV-Flag (Clontech) vectors. These plasmids, including Myc-rig-i, Myc-mda5, Myc-tbk1, Myc-irf3, were constructed using the pCMV-Myc vector as described previously. All constructs were verified by DNA sequencing.
VigoFect (Vigorous Biotechnology, Beijing, China) and FishTrans (MST, FT2020, Wuhan, China) were used for cell transfection. poly I:C (InvivoGen, San Diego, CA) was transfected with Lipofectamine 3000 (Thermo Fisher Scientific). Cycloheximide (CHX), MG132, Baf-A1 and 3-MA were purchased from MedChem Express. NH₄Cl was purchased from Sigma-Aldrich. The antibodies used were as follows: anti-Flag antibody (F1804; Sigma-Aldrich), anti-Myc antibody (9E10; Santa Cruz Biotechnology), anti-HA antibody (MMS-101R; Covance), anti-LC3 antibody (ab48394; Abcam), anti-irf3 (A11921, Abclonal), anti-phosphor-IRF3 (4947, Cell Signaling Technology). anti-ATG5 (A0203, ABclonal), anti-BECN1 (A17028, ABclonal), anti-α-tubulin (62204, Thermo Fisher Scientific), anti-GAPDH antibody (AC033; ABclonal) and anti-β-actin antibody (AC026; ABclonal).