Anti cmyc tag

LPS and CpG-ODN were purchased from Invivogen. DNase I and RNase were purchased from Ambion and Invitrogen, respectively. α-GalCer was purchased from Funakoshi. Mouse Flt3L were purchased from Peprotech. Anti-Flag-tag (SIGMA), anti-cMyc-tag (Clontech), anti-TLR9 (Santa Cruz) and anti-GzmA (rabbit polyclonal) (LSbio) antibodies were from the indicated suppliers. Anti-CD11c (HL3), -CD40 (3/23), -CD80 (6-10A1) and -IFN-γ were purchased from BD. Anti-CD8 (53.67), -CD45.1 (A20), -CD86 (GL-1), -Siglec H(551) and Annexin-V-APC were purchased from Biolegend. Anti-B220 (RA3-6B2) was purchased from e-Bioscience. OVA-tetramers were purchased from MBL. CFSE and Mito-tracker were purchased from Molecular Probes. OVA_257−264 peptide (SIINFEKL) was obtained from Toray Research Center, Inc. Chloropromazine and Latrunculin A were purchased from Wako Co. Diphtheria toxin were purchased from Sigma.

After washed twice with PBS, ICP2 cells were lysed for 30 min on ice, using RIPA lysis buffer (Beyotime, Nanjing, China) with PMSF (Beyotime). Total cell extracts were obtained by centrifugation. Protein concentrations were determined using Compat-Able^TM Protein Assay Preparation Reagent Set (Thermo Fisher Scientific, Shanghai, China). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (Merck-Millipore, Billerica, MA, United States). After blocking with 5% non-fat dry milk, the membrane was incubated with anti-c-Myc tag (1:1000; Clontech, Palo Alto, CA, United States). Anti-β actin (1:1000; Beyotime) was used to ensure protein loading control equally. After incubated with anti-c-Myc tag, the membranes were stripped using the stripping buffer (Beyotime), and re-hybridized with Anti-β actin. The BeyoECL Plus kit (Beyotime) was used for detection.