Clarity western ecl blotting substrate

Protein samples were loaded on 10%, 12% or 15% polyacrylamide gels, depending on the resolving power needed. 15 µg of PBS, TRITON X-100 and NP-40 soluble extracts were loaded for both cell lines (SDS and FA soluble extracts were loaded as equal volume to PBS soluble extracts; insoluble NP-40 extracts were loaded as equal volume to NP-40 soluble extracts).
Proteins were transferred to nitrocellulose membrane (Bio-Rad) with a TransBlot (Bio-Rad) following manufacturer’s instructions. Membranes were then incubated with blocking solution (5% of dried non-fat milk (Euroclone)) for 1 hr at RT and then with primary antibody at 4 °C overnight. After two washes, membranes were incubated with the HRP-conjugated secondary antibody diluted in TBS-T 1X for 1 hr at RT. Signal was detected with ChemiDoc XRS System (Bio-Rad) after incubation with ClarityTM Western ECL Blotting Substrate (Bio-Rad). Antibody description and dilution are described below.

Filter retardation assay (FRA) is based on filtration of proteins on a cellulose acetate membrane with pores of 0.22 µm by a vacuum applied at a Bio-Dot SF Microfiltration Apparatus (Bio-Rad, Hercules, CA, USA). FRA allows the detection of insoluble protein species larger than the pores of the membrane. The following amounts of proteins were loaded: 1.5 µg of NSC34 PBS and NP-40 soluble extracts (NP-40 insoluble extracts were loaded as equal volume), 6 µg of C2C12 PBS and NP-40 soluble extracts (NP-40 insoluble extracts were loaded as equal volume). After vacuum filtration proteins were fixed on the membrane with 20% methanol and then the membrane was incubated for 1 hr at RT in blocking solution (5% of dried non-fat milk (Euroclone) in TBS-T 1X) and then with primary antibody for 1 hr at RT. After two washes with TBS-T 1X the membrane was incubated with the secondary antibody (find incubation timing and antibody dilution below). After three washes with TBS-T 1X at RT membrane was incubated with ClarityTM Western ECL Blotting Substrate (Bio-Rad) to reveal the signal. Images were acquired using a ChemiDoc XRS System (Bio-Rad)^{36 (link)}.

Total cellular proteins from Huh7.0, JFH1-infected Huh7.0, and HCV FL replicon cells were extracted using RIPA buffer (cat. #BP-115, Boston Bioproducts) as previously described. Proteins resolution was done on an 8–15% gel SDS-PAGE denaturing gel. Resolved proteins in acrylamide gels were transferred onto Polyvinylidene fluoride (PVDF) membranes and membranes were blocked in PBS containing 5% dry milk for 1h, then probed with specific primary antibodies in the recommended dilutions at 4°C for overnight. The following primary antibodies were used: anti-HCV NS3 (Abcam cat. #ab13830 and cat. #ab65407); anti-HCV NS5A (Abcam cat. # ab13833 and BioFront cat. # HCV-2F6); anti-HSP90 (Abcam cat. # ab13492); anti-FLAG (Abcam cat. #ab1162); anti-CD63 (Abcam cat. #ab8219 and Santa Cruz Biotechnology cat. #sc-15363); RTN3 antibody (Abcam cat. Ab68328 and Thermofisher cat. # PA5-53360); TSG101 (Abcam cat. # ab125011); normal rabbit IgG-AC antibody (Santa Cruz Biotechnology cat. # sc-2345); anti-beta actin [Ac-15] (Abcam, cat. #ab6276). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology cat. # sc-2004 and sc-2005) and clarity TM Western ECL blotting substrate (Bio-Rad) was used for visualization with the ChemiDoc XRS+ system (Bio-Rad, California, United States).

Cell extracts were prepared in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris–HCl pH 6.8) supplemented with Pierce^TM reducing sample buffer (Thermo Scientific, Waltham, MA, USA). Proteins were resolved by SDS–PAGE using precasted polyacrylamide gels (Biorad, Hercules, CA, USA) and transferred to PVDF membranes (Biorad). Immunoblots were performed using the appropriate primary antibodies and the relative HPR-coupled secondary antibodies (GE Healthcare, Chicago, IL, USA). Proteins were visualized on a ChemiDoc MP imaging system (Biorad) using the ClarityTM Western ECL Blotting Substrate (Biorad). Antibodies are specified in Table 1.

Untreated and stimulated BMMs (1 × 10⁶ cells) were lysed directly in the cell culture vessel using Pierce RIPA buffer (ThermoFisher) supplemented with HALT^TM Protease and Phosphatase Inhibitor (ThermoFisher). Total protein was quantified using the DC assay (Bio-Rad) and resolved on a TGX^TM FastCast^TM Acrylamide gels (Bio-Rad). Gels were imaged directly using the Stain-Free application of a ChemiDoc XR (Bio-Rad) prior to transferring onto a PVDF membrane. Membranes were blocked overnight in 5% non-fat dry milk (w/v), washed and incubated overnight with the appropriate primary antibody. Horseradish peroxidase-conjugated secondary antibodies and Clarity^TM Western ECL Blotting Substrate (Bio-Rad) were used to visualize specified protein bands. Protein densitometry was analyzed according to previously described methodology^{66 (link)}. In brief, band intensity of target proteins was normalized relative to the total protein levels in each respective lane using the Bio-Rad Stain-Free application. Expression of most target proteins were normalized relative to the control sample and presented as a fold change value. Alterations in phosphorylated protein expression were calculated as the increase in phosphorylation as proportion of the total protein. Levels were then compared between the control vs. the treated groups (LPS and PIC) under low and high glucose conditions.

The binding ability of the scFv toward the PEDV S protein was estimated by SEC. First, 380 pmol of PEDV S protein was mixed with 830 pmol scFv and incubated at room temperature for 1 h. The mixture was filtered by a 0.22 μm spin column (Millipore) before being separated by a Superose 6 10/300 GL column (GE Healthcare) in TBA buffer coupled to an AKTA UPC10 FPLC System (GE Healthcare). The protein sample was monitored by the UV absorbance at 280 nm (UV₂₈₀) and fractionated with 0.5 mL per fraction. The fractions that should signify UV₂₈₀ absorbance were analyzed by Western blotting. The proteins were denatured by adding 2 μL of 10× NuPAGE Reducing agent (Invitrogen), 5 μL of 5× NuPAGE Sampling buffer (Invitrogen), and boiled at 95 °C for 5 min. The protein samples were separated by a 10% SDS-PAGE separating gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was blocked with 5% skim milk for 30 min, and probed with 1:5000 diluted anti-V5 tag antibody (Invitrogen) for 1 h at room temperature. After thorough washing, the 1:10,000 diluted anti-mouse IgG antibody with HRP conjugation (Jackson ImmunoResearch Laboratories) was added. After 1 h of incubation and adequate washing, the V5-tag-positive protein signals were detected by using ClarityTM Western ECL Blotting Substrate (Bio-Rad) and visualized by a ChemiDoc XRS+ Imaging System (Bio-Rad).