Donkey anti goat igg

Cells were lysed 72 h after transfection. Cell and tissue samples were lysed in RIRA (Sigma-Aldrich) buffer with PMSF (Sigma-Aldrich). Lysates were centrifuged for 10 min at 12000 × g, and protein in the supernatant was quantified using the BCA method (Pierce). SDS-PAGE and western blot analysis were performed according to standard procedures. Mouse anti-HBs, anti-HBx, anti-HBp, and anti-HBc antibodies were kindly provided by Dr. Quan Yuan from Xiamen University. Goat anti-MAN1A1 (Santa Cruz; 1:500 dilution), rabbit anti-MAN1A2 (Proteintech; 1:200 dilution), rabbit anti-MAN1B1 (GeneTex; 1:500 dilution), mouse anti-MAN1C1 (Abcam; 1:500 dilution) antibodies were used as the primary antibodies. Mouse anti-β-actin (Proteintech; 1:1000 dilution) was used as a reference for protein quantification. Goat anti-mouse IgG (Proteintech; 1:10000 dilution), goat anti-rabbit IgG (Proteintech; 1:10000 dilution), and donkey anti-goat IgG (Santa Cruz; 1:10000 dilution) were used as the secondary antibodies followed by enhanced chemiluminescence (ECL; ThermoFisher Scientific) detection.

Curcumin, glutathione (GSH), N-acetyl cysteine (NAC), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), acridine orange (AO), 3-methyl adenine (3-MA), GW4869, desipramine, phthaldialdehyde (OPA), dimethyl sulfoxide (DMSO), anti-rabbit IgG and anti-mouse IgG were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Oxidation sensitive DCFH-DA (D-399) was from Molecular Probes (Eugene, OR, USA). Dulbecco’s modified essential medium (DMEM), Opti MEM medium, phosphate buffered saline (PBS), trypsin–EDTA and fetal bovine serum (FBS) were from GIBCO BRL (Grand Island, NY, USA). Fumonisin B1, myriocin, and z-VAD-fmk were from Alexis (San Diego, CA, USA). Anti-actin, and anti-MAP LC3β (N-20), anti-p62/SQSTM1, anti-Par-4 and donkey anti-goat IgG antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-PARP, Anti-phospho AMPK Thr¹⁷², Anti-AMPK, Anti-phospho LKB1 Ser⁴²⁸, LKB1, Anti-phospho mTOR Ser²⁴⁴⁸, anti-mTOR , anti-phospho p70S6K Thr³⁸⁹, anti-p70S6K, anti-TFEB, anti-H3 and anti-LC3B (D11) XP antibodies were from Cell Signaling Technology (Beverly, MA, USA). Hydrogen peroxide was from Merck Millipore. MegaTran 1.0 transfection reagent was from OriGene.

One microgram of vesicular proteins was separated by SDS-PAGE and then transferred to a 0.2 µm polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk or 3% skim milk in Tris-buffered saline with 0.05% Tween-20, incubated with primary antibody followed by secondary antibody conjugated with horseradish peroxidase, and subjected to the enhanced chemiluminescence. The membrane was washed three times by Tris-buffered saline with 0.05% Tween-20 after each incubation. Goat anti-actin (1:1000 dilution, SC-1616), rabbit anti-GAPDH (1:1000 dilution, SC-257,780), goat anti-mouse IgG (1:5000 dilution, SC-516,102), goat anti-rabbit IgG (1:5000 dilution, SC-2004), donkey anti-goat IgG (1:5000 dilution, SC-2020) and goat anti-rat IgG (1:5000 dilution, SC-2006) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-CD81 (1:1000 dilution, 555,675) and mouse anti-calnexin (1:1000 dilution, 610,523) antibodies were from BD Biosciences (San Diego, CA). Goat anti-ICAM1 (1:1000 dilution, BBA17) antibody was obtained from R&D Systems (Abingdon, UK). Mouse anti-60 S ribosomal protein L14 (RPL14) (1:1000 dilution, ab89095) antibody was from Abcam (Cambridge, MA). Mouse anti-tubulin (1:1000 dilution, T6074) antibody was from Sigma (St. Louis, MO). Rabbit anti-H2B (1:1000 dilution, 07–371) antibody was obtained from Millipore (Darmstadt, Germany).