Anti mouse igg hrp

Western blotting was conducted according to a protocol previously described [24 (link)]. The antibodies dilution rates were as following: cleaved caspase-3 (ab13585, 2 μg/ml), cleaved caspase-8 (ab25901, 1 μg/ml), cleaved caspase-9 (ab2324, 1 μg/ml), cleaved poly (ADP-ribose) polymerase (PARP) (ab4830, 1:1000), Bcl-2 (ab196495, 1:1000), Bak (ab32371, 1:1000), Bax (ab53154, 1:1000), LC3B-II (ab48394, 1 μg/ml), p62/SQSTM1 (ab56416, 2 μg/ml), Beclin-1 (ab62557, 1 μg/ml), Atg5 (ab228668, 1:1000), p-JNK (ab124956, 1:1000), JNK (ab124956, 1:1000), p-c-jun (ab32385, 1:1000), c-jun (ab32137, 1:1000), Ki-67 (ab15580, 1:1000), GRP78 (ab21685, 1 μg/ml), ATF6 (ab37149), p-eIF2α (ab32157, 1:500), IRE1α (#3294, 1:1000), cleaved caspase-12 (#2202, 1:1000), CHOP (#5554, 1:1000), and cytochrome C (#11940, 1:1000). COX-IV (ab14744, 1:1000), GAPDH (AF1186, 1:1000), IgG-HRP or anti-mouse IgG-HRP (Beyotime, China) (1:3000).

The proteins of PBLC were extracted by using RIPA lysis buffer containing 1% protease inhibitor. After determining protein concentration, a total of 30 μl of samples were separated by 12% SDS-PAGE, transferred to a PVDF membrane and blocked with 7.5% skimmed milk at room temperature for 3 h. The membrane was cropped horizontally according to the target protein and incubated overnight with rabbit anti-IL17A polyclonal antibody (1:1000 dilution, Abcam, Pleasanton, CA, USA) and rabbit anti-BVDV E2 polyclonal antibody (1:2000 dilution, Bioss, Woburn, MA, USA) at 4 °C. Anti-mouse IgG/HRP and goat anti-rabbit IgG/HRP antibody (1: 4000 dilution, Beyotime Biotechnology, Beijing, China) were used as secondary antibodies. Mouse anti-GAPDH and anti-β-actin monoclonal antibody (1:2000 dilution, Beyotime Biotechnology, Beijing, China) were used as an internal standard. The membrane was reacted with ultra-sensitive ECL luminescence reagent (Beyotime Biotechnology, Beijing, China) for color detection. All experiments were performed in triplicate. Grey-scale values of each blot were measured by Image J software, and the intensity of each band was normalized to the loading control GAPDH or β-actin.

XAG (HLPC ≥98%, MW: 392.49) was synthesized as previously described [22 (link)]. Specific antibodies against cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved caspase-12, cleaved PARP, Bcl-2, Bak, Bax, LC3B-II, p62/SQSTM1, Beclin-1, Atg5, p-JNK, JNK, p-c-jun, c-jun, Ki-67, CHOP, GRP78, ATF6, p-eIF2α, IRE1α were purchased from Abcam (Cambridge, UK), specific antibody against cytochrome C was obtained from Cell Signaling (Danvers, MA, USA). Antibody against COX-IV was purchased from Abcam (Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and goat anti-rabbit immunoglobulin horse radish peroxide (IgG-HRP) or anti-mouse IgG-HRP were obtained from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Fluorescent antibody against LC3 was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China).