Semi dry transfer cell

Western blot was performed using Mini-PROTEAN^® Tetra Cell Systems (Bio-Rad) on 10% SDS-PAGE. Proteins were electroblotted onto polyvinylidine difluoride (PVDF) membranes (Immobilon, Millipore) by semi-dry transfercell (Bio-Rad). Membranes were incubated with PTEN (D4.3) XP^® Rabbit monoclonal antibody (Cell Signaling) or PDCD4 (D29C6) XP^® Rabbit monoclonal antibody (Cell Signaling) or Phospho-Akt (Ser473) antibody (Cell Signaling) or Akt (C67E7) Rabbit monoclonal antibody (Cell Signaling) or phospho-IKKα/β (Ser176/180) (16A6) Rabbit monoclonal antibody (Cell Signaling) or phospho-IκBα (Ser32/36) (5A5) mouse monoclonal antibody (Cell Signaling) or NF-κB p65 (L8F6) mouse monoclonal antibody (Cell Signaling) or IKKα/β (H-470) rabbit polyclonal antibody (Santa Cruz) or IκB-α (C-21) rabbit polyclonal antibody (Santa Cruz) at 1:1,000 dilution, or phospho-NF-κB p65 (Ser536) (93H1) Rabbit monoclonal antibody (Cell Signaling) or MMP-9 (D6O3H) XP^® Rabbit monoclonal antibody (Cell Signaling) at 1:500 dilution, or beta-actin specific antibody (Sigma-Aldrich) at 1:10,000 dilution at 4°C overnight. Signals were developed using chemiluminescence Plus Western Blotting Detection System (Amersham) and analyzed using ImageJ software version 1.48 (http://rsbweb.nih.gov/ij/).

The AW ES antigens (20 μg) were subjected to sodium dodecyl sulfat-polyacrylamide gel (SDS-PAGE) in 5% stacking gels and 12% resolving gels at 80 V for 40 min and 120 V for 90 min. After electrophoresis, one gel was stained in Coomassie brilliant blue R-250 staining solution (Sigma, United States) for 4 h, and the other gel was used for the electrotransfer of proteins on nitrocellulose (NC) membranes (Millipore, United States) at 18 V for 35 min via a semi-dry transfer cell (Bio-Rad, United States) (Wang B. et al., 2013 (link)). Subsequently, the membranes were cut into strips, blocked with 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.6) at 37°C for 1 h, and incubated at 4°C overnight with sera from different patients with trichinellosis at a dilution of 1:100. After being washed in TBST, the strips were incubated with HRP-conjugated goat anti-human IgG (1:5,000 dilutions; Sigma, United States) at 37°C for 1 h. Finally, the immunoreaction was detected with 3, 3-diaminobenzidine tetrahydrochloride (DAB; Sigma, United States). The gel and membranes were scanned using ImageScanner (GE healthcare, United States) and the MW of these bands was analyzed by AlphaView software (ProteinSimple, Santa Clara, CA, United States).

For protein isolation, cells were grown in well plates up to 100% confluence. Cells were lysed in RIPA buffer supplemented with protease inhibitors (cOmplete EDTA-free, Roche). Protein quantification was done using a Pierce BCA assay kit (Thermo Fisher Scientific) following the manufacturer’s manual. 25 ug protein per lane (or as stated in the figure legend) were resolved by 8% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham) using a semi-dry transfer cell (BioRad). Membranes were blocked in Rockland blocking buffer (Rockland), incubated with primary antibodies mouse-anti-β-actin (1:2000, Sigma Aldrich, A5316), goat-anti-ACE2 (1:200, R&D, AF933) or rabbit-anti-ACE2 (1:500, abcam, ab15348) and subsequently with IRDye-conjugated secondary antibodies (life technologies) in 5% BSA in TBS containing 0.1% Tween20. Membranes were imaged using an Odyssey IR reader (LI-COR). Quantification was done using Image Studio Lite (LI-COR). The background corrected signal of the ACE2 band at approximately 130 kDa was normalized to the signal of the β-actin band and subsequently to the relative band intensity of a Calu-3 cell control sample.
To study the effect of type I interferon signalling on ACE2 expression in EECM-BMECs, recombinant IFN-α (R&D) at 1 or 0.1 ng/mL was supplemented in hESCR1 medium or SMLC-conditioned medium for 20 h prior to cell lysis.

The protein was extracted from cultured NSC34-G93A cells using Laemmli SDS loading buffer and heated for 8 min at 90 °C. Protein samples were resolved via SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a PVDF membrane with a Bio-Rad semidry transfer cell (1703940, Plano, TX, USA). Membranes were blocked with 5% milk in TBS with 0.1% Tween-20 for 1 h. The primary antibodies used in this study were PGC1α (Abcam ab110324), MT-CO1 (CST 62101S), MT-CO2 (Proteintech 55070-1-AP), COX4 (CST 4844S), and Actin (CST 4970S). After overnight incubation with primary antibody at 4 °C, the membranes were washed with TBST buffer and then incubated with horseradish-peroxidase-conjugated secondary antibody at room temperate for 2 h. Protein bands were visualized using a Bio-Rad Clarity ECL kit under the ChemiDoc Imaging System (Bio-Rad Laboratory). The band intensity was analyzed using ImageJ software.

The recombinant baculovirus-infected Sf9 cells were collected 48 h post infection (hpi). The cell lysates were re-suspended in equal volumes of a 2× Laemmli sample buffer (Bio-Rad, USA), separated by 12% SDS-PAGE, and transferred onto nitrocellulose membranes using a semi-dry transfer cell (Bio-Rad, USA). The membranes were then blocked with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20) containing 5% skim-milk powder. The recombinant VP1 (rVP1) was detected by Western blotting, using an anti-6X His tag^® monoclonal antibody (Abcam, USA) or chicken anti-CAV polyclonal antibodies as the primary antibodies. The expression of rVP2 was also verified by polyclonal antibodies recognizing E. coli-expressed VP2 as the primary antibody. In addition, the detection of recombinant chIL-12 by Western blotting was performed with anti-6X His tag^® monoclonal antibody (Abcam, USA) or polyclonal antibodies recognizing E. coli-expressed chIL-12 as the primary antibodies [27 (link)]. Alkaline phosphatase-conjugated goat-anti-mouse or -chicken IgG (KPL, USA) were used as the secondary antibodies to label the protein bands.