Hrp conjugated goat anti mouse igg

The protein expression levels were analyzed by western blotting as described in our previous studies (Park et al., 2020 (link)). Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell signaling #4022), MMP-9 (Cell Signaling #13667), phospho-PI3K (Cell Signaling #4228), total PI3K (Cell Signaling #4292), phospho-Akt (Cell Signaling #4060), total Akt (Cell Signaling #4491) and β-actin (Abcam, MA, United States, ab189073) overnight at 4°C and then with polyclonal HRP-conjugated goat anti-mouse IgG (BD Pharmingen, 554002) or goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, United States, 554021) secondary antibodies at room temperature for 60 min. The antigen-antibody complexes were detected using Western blot ECL reagents (GE Healthcare, Bucks, United Kingdom).

An ELISA-based assay was also used to determine the C1q binding activity of TsPmy and its fragments according to the method as previously described [28 (link)]. Briefly, a 96-well ELISA plate was coated with human C1q at 5.0 ug/ml in carbonate buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6), blocked with 5.0% (w/v) BSA in PBS at 37 °C for 2 h, then incubated with different concentrations of recombinant TsPmy fragments at 25 °C for 1 h. Anti-His mAb at 1:1000 in PBS/1% BSA was used to probe recombinant proteins, and HRP-conjugated goat anti-mouse IgG (BD Biosciences, Franklin Lakes, NJ, USA; 1:5000 in PBS/1% BSA) was used as secondary antibody. For the peptide binding assay, synthesized peptide P2 was labeled with biotin and the C1q coated plate was incubated with different concentrations of biotinylated peptide P2 (B-P2). HRP-conjugated streptavidin (Abcam, Cambridge, UK; 1:10,000 in PBS/1% BSA) served as the detection antibody. After adding the substrate o-phenylendiamine dihydrochloride (OPD, Sigma-Aldrich, St. Louis, MO, USA), absorbance was measured at 450 nm with a plate reader (Thermo Fisher, Life Technologies). BSA coated on the same plate was used as a negative control.

To compare the binding affinity of TsCRT and its fragments to C1q, 96-well plates were coated with different amounts of human C1q (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, and 1.5 µg/ml) (Millipore, Darmstadt, Germany) in 100 μl/well of coating buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6) overnight at 4°C. The same amounts of BSA were coated in the control wells. Wells were then washed with PBS + 0.05% Tween-20 (PBST) three times, followed by blocking with 200 µl 3% BSA in PBS at 37°C for 1 h. After further washing, 100 µl of 40 nM rTsCRT, rTsCRT-NP, rTsCRT-P, rTsCRT-S, and rTsCRT-C in binding buffer (100 µl of 20 mM Tris–HCl, pH 7.4, 50 mM NaCl, and 1 mM CaCl₂) was added to each well and incubated for 1 h at room temperature. Mouse anti-His mAb (1:10,000, Tiangen, Beijing, China) and HRP-conjugated goat anti-mouse IgG (1:10,000, BD Biosciences, Franklin Lakes, USA) were used as primary and secondary antibodies, respectively. After washing with PBST, the substrate, o-phenylenediamine dihydrochloride (Sigma, St. Louis, MO, USA) was added; colorimetric detection was performed using TMB and the absorbance measured at 450 nm using an ELISA reader (Thermo Fisher).