Peroxidase conjugated anti goat igg

The following antibodies were used in this study: anti-HP1α (MBL; BMP001), anti-HP1β (1MOD-1A9; Millipore), anti-HP1γ (2MOD-1G6; Millipore), anti-DmHP1a (sc-26950; Santa Cruz), anti-Swi6 (41 (link)), peroxidase-conjugated anti-histone H3 (ab21054; Abcam), anti-CK2α (1AD9; Calbiochem), anti-α-tubulin (T5168; Sigma), peroxidase-conjugated anti-FLAG M2 (A8592; Sigma), peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; A6667; Sigma), peroxidase-conjugated anti-mouse IgG (112–035- 072; Jackson ImmunoResearch) and peroxidase-conjugated anti-goat IgG (A5420; Sigma).

Initially, the proteolytic activity of purified Sat was tested against the following purified complement proteins (Complement Technology, USA): C1q, C2, C3 and C3b, C4 and C4b, C5, C6, C7, C8 and C9. To identify possible Sat substrates among these complement proteins, 5 µg of Sat were incubated with 0.5-1.0 µg of each complement molecule in the presence of MOPS buffer (0,1 M MOPS, 0,2 M NaCl and 0,01 mM ZnSO4, pH 7,3) (40 (link)) at 37°C for 5 or 24 h. As a control for spontaneous cleavage, complement molecules diluted in MOPS buffer were incubated under the same conditions. Incubation products were analyzed by immunoblotting using specific antibodies to each complement protein (Complement Technology, USA), and peroxidase-conjugated anti-goat IgG as the secondary antibody (Sigma-Aldrich). Signal detection was performed using the SuperSignal^® West Pico Enhanced Chemiluminescent Substrate (ThermoFisher Scientific) and the Alliance Image System (UVITEC, UK).
Dose dependency of Sat-induced cleavage of the substrates was evaluated using lower concentration of purified Sat (0.5 or 1.0 µg). Also, inhibition of Sat proteolytic activity was assessed by incubating purified Sat (0.5 or 1.0 µg) with 1.0 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min at room temperature before the addition of complement proteins. Incubation products were analyzed as described above.

To analyze whether the proteins of the PDH complex are able to degrade fibrinogen, an assay was performed as reported [30 (link)]. Recombinant proteins rPDHA-D, total proteins of M. pneumoniae or BSA (10 μg/ml of each) were immobilized in wells of a 96-well plate as described. Human plasminogen (10 μg/ml PBS) was added for 2 hours at 37°C. After washing, human uPA (80 ng/ml) and human fibrinogen (20 μg/ml, plasminogen-depleted, Millipore, # 341578) was incubated at room temperature. After different time points, aliquots were taken and the reaction was stopped by heating for 10 minutes at 56°C. The heat-inactivated samples were used to coat 96-well plates overnight at 4°C. After washing and blocking, the degradation of fibrinogen was analyzed using goat anti-fibrinogen (1:1000, Sigma, #F8512) for 90 minutes at room temperature. Detection was carried out with peroxidase-conjugated anti-goat IgG (1:1000, Sigma, #A4174).