Microcon 10kda centrifugal filter unit

The N13 murine microglia cell line (Righi et al., 1991 (link)) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), supplemented with 10% foetal bovine serum and 50 U/ml penicillin/streptomycin (Thermo Fisher Scientific). Cells were maintained in T75 flasks at 37°C in a 5% CO₂ humidified atmosphere. Cells were plated at a density of 2 × 10⁵ cells/cm² in 6-well plates and cultured overnight to allow adherence. Cells were kept in serum-free medium for 4 h prior to stimulation and then incubated without or with 0.1, 1, 10, 100 or 1000 nM of JNJ-527 for 30 min. Recombinant CSF1 (100 ng/ml, R&D Systems) was added to respective wells for 5 min, after which cells were immediately lysed in RIPA buffer (Thermo Fisher Scientific), supplemented with protease and phosphatase inhibitor cocktails (Roche, Thermo Fisher Scientific). Protein lysates were concentrated using Microcon-10 kDa Centrifugal Filter Units (Merck Millipore), according to manufacturer’s instructions and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). For estimation of IC50, values for CSF1R and ERK1/2 phosphorylation were modelled in a non-linear regression curve using GraphPad prism.

The N13 murine microglia cell line (21 (link)) was cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum and 50 U/mL penicillin/ streptomycin (Thermo Fisher Scientific). Cells were maintained in T75 flasks at 37°C in a 5% CO₂ humidified atmosphere. Cells were plated at a density of 2 × 10⁵ cells/cm² in 6-well-plates and cultured overnight to allow adherence. Cells were plated at a density of 2 × 10⁵ cells/cm² in 6-well-plates and cultured overnight to allow adherence. Cells were kept in serum-free medium for 4 h prior to stimulation and then incubated for the indicated time points (5 or 10 min) with recombinant CSF-1 (50 or 100 ng/mL), IL-34 (50 or 100 ng/mL) (R&D Systems) or LPS (1 μg/mL) as a negative control for CSF1R pathway activation (22 (link), 23 (link)), after which cells were immediately lysed in RIPA buffer (Thermo Fisher Scientific), supplemented with protease and phosphatase inhibitor cocktails (Roche, Thermo Fisher Scientific). Protein lysates were concentrated using Microcon-10kDa Centrifugal Filter Units (Merck Millipore), according to manufacturer's instructions and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were subjected to SDS-PAGE and Western blot.

Tumor tissues were mechanically homogenized in lysis buffer (2% SDS, 100 mM Tris-HCl, pH 7.8) supplemented with protease inhibitors (Complete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail, Roche, USA), using a POLYTRON® PT 1200 and sonicated for three cycles at 30% amplitude (20 s bursts with 20 s pauses). Samples were centrifuged at 16,000 x g for 10 min at 4 °C to remove the tissue debris. The supernatants were collected and the protein concentration was measured by using a BCA protein assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific, USA). Aliquots of SDS-lysates containing 200 µg of total protein from each sample were processed according to the filter-aided sample preparation (FASP) method, using Microcon 10 kDa centrifugal filter units (Merck, USA) operated at 10,000 x g for 50 min at 20 °C^{96 (link)–98} . Next, trypsin/LysC Mix (Promega, USA) was added to the filters at an enzyme-to-protein ratio of 1:100 (w/w), and incubated for 12 h at 37 °C; a second digestion was carried out with trypsin (Promega, USA) at an enzyme-to-protein ratio of 1:100 (w/w) at 37 °C for 4 h. Following protein digestion, peptides were filtered through the membrane and purified with reversed-phase chromatography using C18 micro-pipette tips (TopTip^TM, PolyLC inc, USA), according to the manufacturer’s instructions. Peptides were dried in a vacuum concentrator and stored at −20 °C.