Complete ultra tablets and phosstop

RAW 264.7 cells at 1 × 10⁶ cells/cm² were seeded into 12-well plates. The next day, cells were treated with ST2 lysates for 30 min and then exposed to LPS for another 30 min. Cell lysates were prepared using SDS buffer containing protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany). Cell lysates were subjected to SDS-PAGE and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked and the first antibodies bound: NFκB-p65 antibodies (IgG, 1:1000, CST, #8242) and phospho-NFκB-p65 antibody (IgG, 1:1000, CST, #3033), p38 (IgG, 1:1000, SC-#535) and phospho-p38 antibody (IgG, 1:1000, SC, #4511), which were identified with the second antibody labelled with HRP anti-rabbit (IgG, 1:10,000, CST, #7074). Following incubation with Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), chemiluminescence signals were envisioned with the ChemiDoc imaging system (Bio-Rad Laboratories).

Gingival fibroblasts were seeded at 30,000 cells/cm² into 6-well plates. The following day, the medium was changed to serum-free medium overnight. The wells containing PRF were stimulated with 30% PRF overnight, and the following day, the supernatant was applied to the serum-starved cells for one hour with or without inhibitors SB431542 and LDN193189. Subsequently, all cells were stimulated for one hour. Extracts containing SDS buffer with protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany) were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked, and the binding of the first phospho-Smad1/5 (Ser463/465) antibody (CS-9516, anti-rabbit IgG, 1:1000, Cell Signaling Technology) and actin (SC-47778, anti-mouse IgG, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) was detected with the appropriate secondary antibody labeled with HRP (IgG, 1:10,000, Cell Signaling Technology). After exposure to the Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), chemiluminescence signals were visualized with a ChemiDoc imaging system (Bio-Rad Laboratories).

ST2 cells were seeded at 5 × 10⁵ cells/cm² into 12-well plates. The following day, serum-starved cells were exposed to 20% LiPRF, LiPPP, LiBC and LiClot for 30 min and then exposed to TNFα and IL1β for another 30 min. Extracts containing SDS buffer with protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany) were separated by SDS–PAGE and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked, and the binding of the primary antibody phospho-JNK, phospho-p38, phospho-NF-κB p65 and NF-κB p65 (anti-rabbit IgG, 1:1000, Cell Signaling Technology, #4668S, #4511S, #3033, and #8242S respectively) and JNK and p38 (anti-rabbit IgG, 1:1000, Santa Cruz, #sc-474 and #sc-535 respectively) were detected with the secondary antibody labeled with HRP (anti-rabbit IgG, 1:10,000, Cell Signaling Technology, #CS-7074). After exposure to Clarity Wes tern ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), chemiluminescence signals were visualized with a ChemiDoc imaging system (Bio-Rad Laboratories). For densitometric analysis of blots, the WB images were analyzed using Image Lab software (Bio-Rad Laboratories).

RAW 264.7 cells with number of 1 × 10⁶ cells/cm² were seeded into 12-well plates. The next day, cells were treated by ADL for 30 min and then they were exposed to LPS for another 30 min. Lysates of the cells were prepared using SDS buffer containing protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany). Followed by that, lysates were divided by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene Fluoride (PVDF) membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked and the binding of the first antibody NF-κB p65 antibodies (IgG, 1:1000, Cell Signaling Technology, #8242, Cambridge, UK), phospho-p65 antibody (IgG, 1:1000, Cell Signaling Technology, #3031), was identified with the second antibody labelled with horseradish peroxidase anti-rabbit (IgG, 1:10,000, Cell Signaling Technology, #7074). Following incubation by the Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) chemiluminescence signals were envisioned with the ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

RAW 264.7 cells were seeded at 50,000 cells/cm² into 6-well plates. The following day cells were exposed to 10% of PPP, BC, Alb-gel and RC for 30 minutes and then they were exposed to LPS for 40 minutes. Extracts containing SDS buffer with protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany) were separated by SDS-PAGE and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked and the binding of the first antibody NF-κB p65 antibodies (IgG, 1:1000, Cell Signaling Technology, #8242), phospho-p65 antibody (IgG, 1:1000, Cell Signaling Technology, #3031), was detected with the second antibody labelled with HRP anti-rabbit (IgG, 1:10,000, Cell Signaling Technology, #7074). After exposure to the Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA) chemiluminescence signals were visualized with the ChemiDoc imaging system (Bio-Rad Laboratories). For blot densitometric analysis images were analyzed using Image Lab software (Bio-Rad Laboratories).

ST2 cells were seeded at 5 × 10⁵ cells/cm² into 12-well plates. The following day, serum-starved cells were exposed to 10% of PPP, BC, Alb-gel, and RC or 30% of PRF lysates for 30 min, and then they were exposed to TNFα and IL1β for another 30 min. Extracts containing SDS buffer with protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany) were separated by SDS-PAGE and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked, and the binding of the first antibody phospho-NF-κB p65 antibodies (anti-rabbit IgG, 1:1000, Cell Signaling Technology) and NF-κB p65 antibody (1:1000, Cell Signaling Technology) was detected with the second antibody labelled with HRP (CS-7074, anti-rabbit IgG, 1:10,000, Cell Signaling Technology). After their exposure to the Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), chemiluminescence signals were visualized with the ChemiDoc imaging system (Bio-Rad Laboratories). For densitometric analysis of blots, the WB images were analyzed using Image Lab software (Bio-Rad Laboratories).