Hrp conjugated goat antirabbit a0545

The following antibodies were obtained from Sigma-Aldrich: mouse anti-β-actin (A1978), mouse anti-Flag (F1804), horseradish peroxidase (HRP)-conjugated goat antimouse (A9044), and HRP-conjugated goat antirabbit (A0545). Rabbit polyclonal antibodies against MAVS (A5764) and RNF5 (A8351) were purchased from ABclonal Technology. Rabbit polyclonal antibodies against MARCH5 (ab185054) and HA (3724s) were obtained from Abcam and Cell Signal Technology, respectively. Rabbit anti-N polyclonal antibody and mouse anti-N monoclonal antibody were prepared in our laboratory. The proteasome inhibitor MG-132 (S2619, Sellek), the autophagy inhibitor wortmannin (S2758, Sellek), and Chloroquine (CQ) (C6628, Sigma) were used in the experiments. The Enhanced Cell Counting Kit-8 (C0042) and DAPI (C1002) were purchased from Beyotime.

Protein concentration in the lysates was determined using the BCA assay (Pierce, Rockford, IL, USA) and adjusted with an appropriate volume of Laemmli buffer (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were resolved on a 12% SDS-PAGE gel for 1 h at 160 V. Samples were transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA) for 1 h at 100 V, 400 mA on ice, and subjected to western blot analysis. The membrane was blocked with Tris-buffered saline, 5% (w/v) non-fat dry milk for 30 min and probed overnight at 4 °C with a rabbit RGS2 antibody (a gift from Dr. David Siderovski). The membrane was probed for 1 h with horseradish peroxidase (HRP)-conjugated Goat anti-rabbit (A0545; 1 : 10 000, Sigma-Aldrich, St. Louis, MO, USA) secondary antibody diluted in TBS-T, 5% (w/v) non-fat dry milk. The protein bands were visualized on autoradiography film using the Super Signal West Pico chemiluminescent substrate (Pierce, Waltham, MA, USA).