Enhanced chemiluminescence western blotting kit

SARS-CoV-2 NTU13-infected cell lysates provided by NTUH (28 (link)) were harvested using RIPA buffer III (Bio Basic Inc., Markham, Ontario, Canada). The cell lysates or recombinant proteins were prepared under reducing or nonreducing conditions prior to loading onto 10% or 12% SDS–PAGE gels for separation as indicated. For non-reducing condition, sample buffers containing 2% SDS and 15% glycerol were added to the samples prior to loading into SDS-PAGE; for reducing condition, sample buffers containing 2% SDS, 15% glycerol and 1% 2-ME were added to the samples, following by heat-denaturing at 95°C for 5 min prior to loading into SDS-PAGE. The separated proteins were transferred onto a PVDF membrane (Pall, Ann Arbor, MI, USA). The membrane was blocked with 5% skim milk in TBST (0.05% Tween 20 in Tris-buffered saline, TBS), incubated with primary antibodies, namely, anti-His antibody (10411, Leadgene Biomedical Inc.), anti-SARS-CoV-2 S1 mAb (GTX635656, GeneTex Inc, Irvine, CA, USA), anti-ACE2 polyclonal antibody (anti-ACE2 pAb; ARG41099, Arigo, Taiwan) or mAbs 127 and 150 overnight at 4°C and detected with HRP-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies (1:10,000 dilution; Leadgene Biomedical) for another 1 h. Detection was then performed using an Enhanced Chemiluminescence Western blotting Kit (Advansta, Menlo Park, CA, USA).

Whole-cell lysates were collected using RIPA buffer III (Bio Basic Inc., Markham, Ontario, Canada) and subjected to 12% SDS-PAGE for separation. Next, the gel was transferred onto a PVDF membrane (Pall, Ann Arbor, MI, USA) and blocked with 5% skim milk in TBST (0.05% Tween 20 in TBS). Primary antibodies against LC3 (MBL International, Woburn, MA, USA), phosphor Tyr 705-STAT3 (Cell Signaling Technology, Beverly, MA, USA), total STAT3 (Cell Signaling Technology), BNIP3 (Abcam, Cambridge, MA, USA), caspase 3 (Imgenex, San Diego, CA, USA), β-actin (Sigma-Aldrich, St Louis, MO, USA) or MIF rabbit polyclonal antibody purified from recombinant human MIF (rMIF)-immunized rabbit sera were incubated with membrane at 4 °C overnight and washed with TBST. HRP-conjugated goat anti-rabbit or rabbit anti-mouse IgG secondary antibodies (1 : 10 000 dilution; Leadgene Biomedical) were incubated for another 1 h and detected using an Enhanced Chemiluminescence Western Blotting Kit (Advansta, Menlo Park, CA, USA). The results of the western blotting were quantified using the Image J software (National Institutes of Health, New York, NY, USA).

After behavioral tests, rats were euthanized under anesthesia with isoflurane (Abbott Laboratories Ltd., Queenborough, Kent, UK), and the left dorsal quadrant section of the lumbar spinal cord is isolated and preserved at −80 °C. The tissue sections were homogenized in ice-cold 1X radioimmunoprecipitation assay lysis buffer using a cell disruptor under sonication (Misonix, Inc. Newtown, CT, USA), then the contents were centrifuged at 13,000 RPM for half an hour at 4 °C. The supernatant portion is carefully collected, quantified using Bradford protein assay. The protein denaturing is done by heating at 90 °C for 10 min in an equal volume of reducing sample buffer and separated using 12% SDS-PAGE and proteins were transferred on to a polyvinylidene fluoride membrane (Pall, Ann Arbor, MI, USA) and kept for blocking using 5% skimmed milk in tris-buffered saline (0.05% Tween 20 in tris-buffered saline). Primary antibodies against CD-11B antibody (Genetex Cat No. GTX134493, Alton Pkwy Irvine, CA, USA) were incubated at 4 °C overnight followed by washing with TBST. Furthermore, they were incubated with horseradish peroxidase conjugated goat anti-rabbit antibody (Leadgene Biomedical Cat# 20202, Tainan, Taiwan) for another 3 h and detected using an Enhanced Chemiluminescence Western Blotting Kit (Advansta, Menlo Park, CA, USA).