Srx 101a

Whole cell lysates were collected using RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 2 mM EDTA) and separated by SDS-PAGE on polyacrylamide gel (8–15% acrylamide depending on protein targets). Proteins were transferred to nitrocellulose membranes (Biorad, Hercules, CA, USA) and membranes were blocked in Blocking buffer (5% milk in TBS containing 0.1% Tween-20) for 1 h at room temperature. Membranes were incubated with primary antibody diluted in blocking buffer overnight at 4 °C. Appropriate HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) were used at 1:5,000 dilution in blocking buffer for 1 h at RT. Signals were visualized with BioRad chemiluminescence reagents and either xray films developed with a Konica SRX-101A (Konica Minolta, Wayne, NJ, USA) film processor or with a GeneGnome XRQ NPC system (Syngene, Frederick, MD, USA). Densitometric quantifications were performed with ImageJ (National Institutes of Health, Bethesda, MD, USA). Membranes were cut prior to hybridization with antibodies.

The Proteome Profiler Mouse Cytokine Array Kit (Panel A; R&D Systems, USA) was used to analyze the cytokine arrays using filtered supernatant collected from primary mouse microglial cultures at 48 h incubation in the presence or absence of CSF-1RAb LPS and ATP. Assays were carried out according to the manufacturer’s protocol as previously described (37 (link)). The array membranes were imaged with film developer (SRX-101A, Konica Minolta, USA) and the results were analyzed using ImageJ software.

Cells were collected and resuspended in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was quantified using the Bradford Protein Assay (20 μg was loaded per well). Protein samples were then resuspended in Laemmli buffer and separated on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. Antibodies used included anti-p53 (1:1000), anti-p21 (1:1000), anti-β-actin (1:5000), anti-α-tubulin (1:2000), anti-Histone H3 (1:10000), anti-Interleukin-8 (1:1000), anti-NRF2 (1:1000), anti-ERK (1:1000), anti-pERK Thr202/Tyr204 (1:1000), pEGFR Tyr1068 (1:1000) anti-MCAK (1:1000), anti-N-cadherin (1:500), anti-E-Cadherin (1:500), anti-Vimentin RV202 (1:500), anti-pHER2 Tyr1221/1222 (1:1000) and anti-p-c-Met Tyr1234/1235 (1:1000). Western blots were developed using SRX-101A Konica Minolta and scanned. The intensity of the bands was measured by densitometry using ImageJ (National Institute of Health, Bethesda, MD, USA).
To assess the levels of p-Erk1/2, cells pre-treated with HER2 (Trastuzumab, 40 μg/ml, 1 hr) and CXCR1/2 (SCH563705, 100 nM, 1 hr) inhibitors and incubated with CM for 10 min.

Mouse blood was allowed to clot for 4 h at room temperature. After centrifuging for 15 min at 2,000xg, supernatant (i.e., serum) was carefully obtained and stored at −80^oC until further use. Cytokine array experiment was conducted according to the manufacturer’s protocol (R&D, Minneapolis, MN, cat. No. ARY028). Blots were imaged via chemiluminescence method; X-ray film exposure in a dark room and development with SRX-101A medical film processor (Konica, Tokyo, Japan). Protein levels were quantified using ImageJ software.

Cell lysates and brain homogenates were extracted with RIPA (1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.2) supplemented with cOmplete™ protease inhibitor cocktail (Roche), and equal amounts of protein samples (20–80 μg) were separated in SDS-PAGE gels, followed by immunoblotting with primary antibodies and detected with HRP conjugated secondary in 5 % non-fat dried milk or 5 % bovine serum albumin (BSA) in TBST. Membranes were developed using ECL™ (GE Amersham) reagents and using Hyperfilm ECL™ in an automated developer from Konica, SRX 101A. To re-probe blots for a different protein, membranes were stripped with ReBlot Plus Strong Antibody Stripping Solution (Millipore). Digital images were quantified by densitometry using ImageJ and adjusted for protein loading by normalising to β-actin, GAPDH or tubulin, or full-length APP for APP cleavage products.