Supersignal west pico plus chemiluminescent substrate solution

PDL cells were plated in 60 mm tissue culture plates at 1.5x10^-6 cells/plate overnight. Cells were then challenged with wildtype T. denticola at an MOI of 50 for 2 h or left unchallenged as controls. Post-challenge, cells were washed three times with PBS and incubated in αMEM medium for 24hrs. Cells were mechanically harvested with a cell-scraper, pelleted under centrifugation, washed with PBS, and lysed using radioimmunoprecipitation assay (RIPA) buffer. Lysates were then electrophoretically resolved on pre-made 4-12% bis-tris polyacrylamide gels (Invitrogen) and transferred onto immobilon-P PVDF transfer membranes (EMD Millipore) for western blotting analysis to evaluate changes in actin monomers (β-actin and γ-actin). Primary antibodies included: β-actin (Abcam; ab8227; RRID : AB_2305186), γ-actin (Santa Cruz; Cat# sc-65635, RRID : AB_1120816), and glyceraldehye 3-phosphate dehydrogenase (GAPDH) (Santa Cruz; sc-32233, RRID : AB_627679). Supersignal West Pico Plus Chemiluminescent substrate solution (Thermo Scientific) was used to develop protein bands. Densitometry was performed using FIJI imaging analysis software.

A total of 40 μg of PBMC proteins were separated by molecular weight using a SDS-PAGE, 4%–20% Criterion™ TGX™ Precast gels (cat# 5671095, Bio-Rad, Hercules, CA, USA). The gel proteins were transblotted onto polyvinylidene difluoride (PVDF) membranes and incubated in 5% nonfat dry milk for an hour at room temperature. Further, blots were incubated at 4 °C overnight with a primary antibody against PTX3 (1:5000, cat# ab125007, Abcam, Cambridge, UK), 4-HNE (1:1000, cat# ab46545, Abcam, Cambridge, UK), 3-nitrotyrosine (3NT) (1:1000, cat# 9691, Cell Signaling Technology Inc), TLR4 (1:500, cat# 293072, Santa Cruz Biotechnology) or GAPDH (1:5000, cat# 97166, Cell Signaling Technology Inc) in 5% nonfat dry milk. The protein carbonyls (PC) detection was performed according to manufacturer’s instruction using an OxyBlot kit (cat# S7150; Millipore Inc). For secondary antibodies, peroxidase-conjugated horse anti-mouse IgG (cat# 7076) and goat anti-rabbit IgG (cat# 7074) from Cell Signaling Technology Inc were used. The immunoreactive protein reaction was exposed in ChemiDocTM XRS+ imaging system (Bio-rad) using a SuperSignal™ West Pico PLUS Chemiluminescent substrate solution (cat# PI34580, Thermo Fisher), and band density analysed by the ImageJ software (NIH, Bethesda, MD, USA).