Anti alix 12422 1 ap

Colon tissues, cells, and EVs fractions were homogenized using the RIPA buffer (Jiangsu KeyGEN BioTECH, Nanjing, China), phosphatase inhibitor cocktail, and protease inhibitor cocktail (Jiangsu KeyGEN BioTECH, Nanjing, China). Pierce BCA Protein Assay Kit (Jiangsu KeyGEN BioTECH, Nanjing, China) was used to determine the protein concentration. Anti-CD63 (A5271, ABclonal), anti-Alix (12422-1-AP, Proteintech), anti-CD81 (66866-1-Ig, Proteintech), anti-Calnexin (ab22595, Abcam), anti-STING (19851-1-AP, Proteintech), anti-Phospho-STING (Ser366) (85735, Cell Signaling Technology), anti-Phospho-STING (Ser365) (72971, Cell Signaling Technology), anti-IRF3 (4302, Cell Signaling Technology), anti-Phospho-IRF3 (Ser396) (29047, Cell Signaling Technology), anti-NF-kB p65 (A19653, ABclonal), anti-Phospho-NF-kB p65 (Ser536) (3303, Cell Signaling Technology), anti-Beta-Actin (4970, Cell Signaling Technology), anti-GAPDH (AF1186, Beyotime Biotechnology) and secondary antibodies (KGP1201, Jiangsu KeyGEN BioTECH, Nanjing, China) were used for western blot.

Rabbit polyclonal anti-HBc antibody is from DAKO (B0586). For the detection of capsids, mouse monoclonal anti-HBc C1 (ab8637) was purchased from Abcam, and mouse monoclonal anti-HBc 2A7 was kindly provided by Prof. Quan Yuan (Xiamen University, China). For the immunoprecipitation of HBV virions, rabbit anti-HBs (NB100-62652, Novus, Centennial, CO, USA) and rabbit anti-preS1 antibodies (kindly provided by Prof. Shuping Tong, Brown University, USA) were used. For immunofluorescence staining, commercially available rabbit polyclonal anti-Alix (12422-1-AP, Proteintech, Chicago, IL, USA), and rabbit polyclonal anti-HBc (0282, Long Island Antibody, Shanghai, China) were used. Other commercially available antibodies are as follows: mouse anti-β-tubulin (M20005, Abmart, Shanghai, China), mouse anti-β-actin (T40104, Abmart, Shanghai, China), and rabbit anti-Flag (14793S, Cell Signaling Technology, Boston, MA, USA).

Cells and sEVs were lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 0.1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (cat. no., 25955; Nacalai Tesque). Lysates and immunoprecipitates were denatured in Laemmli buffer containing DTT and 2-mercaptoethanol, respectively. The denaturation was performed for 2 h at 37 °C for the lysates of EVs and for 5 min at 100 °C for the other samples. After cooling, the proteins were separated by SDS-PAGE, transferred onto PVDF membranes, blocked with 5% milk or 2% BSA for 1 h and probed with following primary antibodies: anti-Alix (12422-1-AP; Proteintech), anti-β-actin (clone AC-74; SIGMA), anti-CD9 (ab92726; Abcam), anti-ephrin-A1 (AER-031; Alomone Labs), anti-EphA2 (C-20; Santa Cruz), anti-Erk (#9102; CST), anti-FLAG (PM020; MBL), anti-p21 (C-19; Santa Cruz), anti-phospho-Erk (E-4; Santa Cruz), anti-phospho-p53 (Ser15) (#9284; CST), anti-phosphotyrosine (clone 4G10; Millipore), anti-PTP1B (clone FG6-1G; Millipore) and anti-Rab35 (11329-2-AP; Proteintech). All primary antibodies were diluted to 1 μg ml⁻¹. After washing, membranes were incubated with secondary antibodies (GE Healthcare) (1:1,000) for 1 h at room temperature (RT) and visualized with enhanced chemiluminescence reagents. Uncropped gel images are provided in Supplementary Figs 6–12.