Sigmafast protease inhibitor cocktail

Cell extracts were prepared in RIPA buffer, containing 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% SDS, 1% NP-40, 0.5% sodium deoxycholate and SigmaFAST protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Ten micrograms of proteins were loaded onto the gel in Laemmli buffer and separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA). Non-specific binding sites on the membrane were blocked with 1% casein in TBST (0.1% Tween) for 1 h at room temperature (RT). Then, the membrane was incubated overnight at 4 °C with anti-IκB (1:1000) (Santa Cruz Biotechnology, Dallas, Texas, USA) antibody and/or 1 h at RT with anti-actin-HRP (1:2000) (Santa Cruz Biotechnology) or anti-rabbit-HRP (1:3000) (Dako, Agilent Technologies, Santa Clara, CA, USA) antibodies. HRP-catalyzed reactions were developed using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).

Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma, Taufkirchen, Germany). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in PBS. The following antibodies were used: alpha-Tubulin (Sigma, Taufkirchen, Germany), HOXB5 (Santa Cruz Biotechnology, Heidelberg, Germany), and BCL6 (Cell Signaling Technology, Danvers, MA, USA). For loading control, blots were reversibly stained with Poinceau (Sigma, Taufkirchen, Germany) and detection of alpha-Tubulin (TUBA) performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western Lightning ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).

The selected brain regions were sonicated in a lysis buffer containing 50 mM NaCl, 50 mM HEPES, 2 mM sodium orthovanadate, 1% Triton X-100, and SigmaFAST Protease inhibitor cocktail (Sigma-Aldrich). After quantification and denaturation, the samples were loaded and separated by 12% SDS-PAGE gels and then transferred into a nitrocellulose membrane. The membranes were incubated for 1 h in blocking solution (BS: 5% Bovine serum albumin, 1% Tween 20 in PBS), and incubated overnight at 4°C with primary antibodies to GDNF (1:500 in BS; Abcam ab119473), BDNF (1:400 in BS; Promega G1641), or proBDNF (1:500 in BS; Invitrogen PA1-18360), together with anti-alpha-tubulin (1:3000 in BS; Abcam ab184613) as loading control. Afterward, the membranes were washed and incubated for 1 h at room temperature with IRDye 680RD/IRDye 800CW-Conjugated Goat Anti-Mouse IgG/Goat Anti-Rabbit IgG/Donkey Anti-Chicken IgG secondary antibodies (1:15000 in PBS each, LI-COR Biosciences #926-68070, #926-32210, #926-68071, #926-32211, and #925-32218). The Odyssey system (LI-COR Biosciences) was used to detect the bands. Quantification of band intensity was performed using Image Studio software version 5.2.5.

Whole cell extracts from NIH/3T3 cells and NPCs were prepared in RIPA lysis buffer (50 mM Tris-HCl pH-7.4, 150 mM NaCl, 2% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1mM DTT, 10% glycerol, 1x SIGMAFAST protease inhibitor cocktail (Sigma-Aldrich), and 1x PhosSTOP (Roche)). Samples were resuspended in NuPAGE-LDS sample buffer (Thermo Fisher Scientific), incubated at 37°C for 30 min, and subjected to SDS-PAGE. The resolved proteins were transferred onto a nitrocellulose membrane (Bio-Rad) using a wet electroblotting system (Bio-Rad) followed by immunoblotting.

DPSCs and PDLSCs at passage 3 were seeded into 90 mm Petri dishes (Eppendorf AG, Hamburg, Germany) and cultured in standard conditions with DMEM (ThermoFisher Sci, Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 37 °C, 5% CO₂. When cells reached 90–100% confluency, the medium was changed to osteogenic medium prepared ex tempore (Low glucose DMEM supplemented with 10% FBS, 2 mM l-glutamine, 1% penicillin/streptomycin (HyClone, Logan, UT, USA), 50 mg/mL ascorbic acid (Sigma Aldrich, USA), 0.1 mM dexamethasone (Sigma Aldrich, USA) and 10 mM β–glycerophosphate (Sigma Aldrich, St. Louis, MO, USA).
Control and differentiated (10th day after induction of osteogenic differentiation) cells were lysed by RIPA lysis buffer (ThermoFisher Sci, Waltham, MA, USA) with SIGMAFAST protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) then the lysates were collected to microcentrifuge tubes, frozen, sonicated and centrifuged (15 min, 16,000× g, 4 °C).