Westernbright ecl detection kit

Cells were lysed in RIPA buffer (Boston Bioproducts) supplemented with SigmaFast protease inhibitors (Sigma Aldrich). Protein samples (20 µg) were separated on 10% SDS–polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Immunoblotting was performed using rabbit polyclonal antibodies directed against mouse UBASH3A at 4 ºC overnight, followed by goat anti-rabbit secondary antibody (Millipore, 12–348) at room temperature for 45 min. The rabbit anti-UBASH3A polyclonal antibody was produced by Thermo Scientific using the oligopeptide EKLQEFWRESRRQCAKNR (amino acid residues 86–103) as an antigen. Binding of the secondary antibody was detected by the Western bright ECL detection kit (Advansta Corporation). The membrane was then stripped with Restore plus Western Blot stripping buffer (Thermo Scientific) and re-probed with the rabbit anti-α tubulin polyclonal antibody (Abcam, ab4074).

Total proteins were extracted with 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 % NP40, and Roche phosphatase and protease inhibitor cocktail. Immunoprecipitation was performed overnight at 4 °C on total protein lysates with the indicated antibodies according to the supplier’s recommendations. Protein G sepharose beads were then added for 1 hour at 4 °C. Proteins were eluted with 50 μl loading buffer (45 μl LDS sample buffer + 5 μl Reducing Agent; Novex, ThermoFisher Scientific, Waltham MA, USA,) and then boiled at 95 °C for 5 minutes. Immunoprecipitates or 50 μg of proteins for a whole cell lysate were loaded onto 4–12 % SDS-PAGE gels using the NuPAGE system from Invitrogen (ThermoFisher Scientific, Waltham MA, USA) (30 mA for 10 minutes and 50 mA for 1 hour 15 minutes) and then transferred onto Invitrolon PVDF membranes in the Biorad Blotter system (Hercules CA, USA), using blotting buffer containing 25 mM Tris-Base, 192 mM glycine, and 5 % methanol (100 V for 30 minutes). Detection was by chemiluminescence using the Western Bright ECL detection kit Advansta (Menlo Park CA, USA). Immunoblots are representative of a minimum of three independent experiments. An equal amount of protein was loaded onto each gel and the immunoblots shown in the same figure were developed simultaneously with the same exposure time.

We used the Yuliang Wu far‐western blotting method³⁵; 20 μg of all purified proteins (RBD, hCypA, and ACE2) was loaded into wells with 62.5 mM Tris–HCl pH 6.8, 10% glycerol, 1% SDS, 1% β‐mercaptoethanol and 0.01% bromophenol blue for 5 min at 95°C. Total proteins were separated using 4%–12% SDS‐PAGE at 120 mA for 2 h and transferred to a Polyvinylidene Fluoride (PVDF) membrane (Amersham, USA) at 100 V for 2 h. The membrane was stained with Coomassie Brilliant Blue and Ponceau S (Sigma, USA) to determine whether the proteins had transferred from the gel to the membrane. Next, the proteins were denatured and refolded on the membrane in the AC buffer (100 mM NaCl, 20 mM Tris–HCl pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween‐20, 2% skim milk, and 1 mM DTT) by gradually reducing the guanidine–HCl concentration. The membrane was then blocked with 5% (w/v) blocking agent (GE Healthcare, USA) for 1 h at RT and incubated with 10 μg purified “bait” RBD protein RBD in PBS overnight at 4°C. The membranes were washed five times with PBST buffer (PBS containing 0.1% [v/v] Tween 20) and incubated with anti‐RBD antibody for 2 h at 4 °C. After incubation, the membrane was washed with PBST buffer three times and probed with an anti‐rabbit secondary antibody (Sigma, USA) for 1 h at RT. Immunoreactive proteins were detected using a WesternBright ECL detection kit (Advansta, USA).

To verify the glycoprotein observed in label-free relative quantification, immunoblot analyses of complement C9 (C9) and fibronectin (FN1) were performed. Equal amounts of protein from non-metastatic CRC, metastatic CRC, and healthy control groups of crude plasma, WGA-bound and WGA-unbound fractions were separated by 10% TGX Stain-Free FastCast. The total protein levels of each sample were visualized using Gel Doc™ EZ Imager and then transferred onto PVDF membranes. The membranes were blocked in 3% bovine serum albumin (BSA) in TBS/T for 1 h and probed with primary antibodies; anti-complement C9 (ab173302, Abcam, Cambridge, UK) and anti-fibronectin (ab32419, Abcam, Cambridge, UK) at 4 °C overnight. After washing, the membranes were incubated with an anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (P0217, Dako, Glostrup, Denmark) in 5% skim milk in TBS/T at RT for one hour. The signal on the membrane was visualized using WesternBright ECL detection kit (Advansta, San Jose, CA, USA) and captured by ImageQuant LAS 4000 digital imaging system (GE Healthcare, Piscataway, NJ, USA). The band intensities of C9 and FN1 were normalized to its stained total protein intensity on the gels for each sample.