Signalfire plus ecl reagent

Cells were lysed on ice in a lysis buffer composed of 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) and 5 mM EDTA with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 500 g for 30 min at 4 °C to remove cellular debris. Proteins (25 μg/lane) were separated on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were immunoblotted for CSF1R (1:250; #MAB3291, R&D Systems) and its phosphorylated form (Y723; 1:500; #3155, Cell Signaling), nuclear factor kappa B (NF-κB; 1:500; #8282, Cell Signaling) and its phosphorylated form (S536; 1:500; #3033, Cell Signaling), caspase-1 (1:500; #ab179515, Abcam), IL-1β (1:500; #12,242, Cell Signaling), NLR family pyrin domain containing 3 (NLRP3; 1:500; #15101S, Cell Signaling) and GAPDH (1:5000; G8795, Sigma Aldrich) overnight at 4 °C, and then with horse radish peroxidase-linked secondary antibodies (1:10,000; Jackson Laboratory) for one hour. Bands were detected by enhanced chemiluminescence SignalFire™ Plus ECL reagent (Cell Signaling) using a ChemiDoc Imaging System (Bio-Rad Laboratories). Image analysis was performed using ImageLab 6.0.1 software (Bio-Rad Laboratories).

The cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Delaware, CA, USA) containing a 1x Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The protein content was measured with a protein assay kit (Pierce, Hercules, CA, USA). Protein samples (15 μg), together with a protein marker (Precision Plus Protein Western C Standards; Bio-Rad), were separated on 12% Mini-Protean TGX gels (Bio-Rad, Richmond, CA, USA) for 30 min at 200 V. The separated gels were transferred to a polyvinylidene fluoride (PVDF) membrane for 3 min using the Trans-Blot Turbo Transfer system (Bio-Rad) with Trans-Blot Transfer Packs. Western blots with Runx2), ALP, OSX, and β-actin (ACTB) were processed on the iBind Western System (Life Technologies, Carlsbad, CA, USA) as specified by the antibody manufacturer (anti- Runx2) [Abcam, Tokyo, Japan], anti-ALP [Abcam], anti-Sp7/osterix [OSX; Abcam], and anti- ACTB [Bio-Rad] primary antibodies; and horseradish peroxidase-conjugated anti-mouse secondary antibody [Bio-Rad]). The incubated membranes were developed using an enhanced chemiluminescence system (SignalFire Plus ECL Reagent; Cell Signaling Technology). The band density was quantified using the NIH-Image J software and normalized to that of ACTB on day 0.

BMDE were cocultured with A. fumigatus conidia or left unstimulated for 1 h. Cells were lysed with RIPA buffer including cOmplete protease inhibitor and PhosSTOP phosphatase inhibitor (both Roche, Basel, Switzerland). Protein extracts were denatured by boiling in Laemmli buffer for 5 min, run on an SDS-PAGE, and then blotted semidry onto a polyvinylidene difluoride membrane. Membranes were soaked in 3% bovine serum albumin (BSA) in TRIS-buffered saline-Tween buffer (TBS-T) for 1 h at room temperature and then incubated with the primary antibody overnight at 4°C. Antibodies against phosphorylated (D9E) and total AKT (C67E7), as well as β-actin (13E5) (all Cell Signaling Technology), were applied. Horseradish peroxidase (HRP)-linked polyclonal goat anti-rabbit antibody (Rockland Immunochemicals, Limerick, PA) was used as a secondary antibody for 1 h, followed by development in SignalFire Plus ECL reagent (Cell Signaling Technology) and luminescence imaging on a Bio-Rad ChemiDoc imaging system.

Molecular weight marker, reagents and nitrocellulose membranes for SDS‐PAGE were purchased from Bio‐Rad (Mississauga, ON). SignalFirePlus ECL reagent (catalogue #12630), protease/phosphatase inhibitor cocktail (catalogue #5872), and SDS loading buffer/dithiothreitol (catalogue #7722) were from Cell Signaling Technologies (Danvers, MA). Horseradish peroxidase–conjugated donkey anti‐rabbit and goat anti‐mouse IgG secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibodies against BACE (#5606), ERK (#4695), phospho ERK (threonine 202/tyrosine 204; #9101), p38 (#9212), phospho p38 (threonine 180/tyrosine 182; #9211), JNK (#9252), phospho JNK (threonine 183/tyrosine 185; #4671), GAPDH and VINCULIN were purchased from Cell Signaling (Danvers, MA).

Immunoblotting has been described previously (1 (link)). Primary antibodies used include: SIRPα (1:1,000; #13379; Cell Signaling), phospho-Shp1 (1:1,000; Tyr564; #8849; Cell Signaling), total Shp1 (1:1,000; #3759; Cell Signaling), phospho-Shp2 (1:1,000; Tyr580; #3703; Cell Signaling), total Shp2 (1:1,000; #3397; Cell Signaling), phospho-Lck (1:1,000; Y394; #ab201567; Abcam), total Lck (1:1,000; #2752; Cell Signaling), phospho-SYK (1:1,000; Tyr525/526; #2710; Cell Signaling), total SYK (1:1,000; #13198; Cell Signaling), phospho-AKT (1:1,000; Ser473, #5082; Cell Signaling), total AKT (1:1,000; #9272; Cell Signaling), GAPDH (1:5,000; #5174; Cell Signaling), and β-actin (1:5,000; sc47778; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; Cell Signaling) and goat anti-mouse secondary antibodies (1:5,000; Sigma-Aldrich) were used for detection. SignalFire™ Plus ECL reagent (#12630S, Cell Signaling) was used for protein expression detection as per the manufactures protocol. Fusion SL (Analis Instruments) was used as per manufacturer instructions and chemiluminescent captured using Fusion SL (Analis Instruments). Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or β-actin or GAPDH using Image J software.

Cell lines (22Rv1, VCaP, LNCaP, and PC3) were cultured to 70% confluency, harvested in urea lysis buffer (9.8 M Urea, 15 mM EDTA, 30 mM Tris) and homogenized by ultrasonic treatment. Protein concentration was measured with the Pierce BCA Protein Assay Kit (Pierce, Rockford, Illinois, USA). 40 µg of total protein was applied for Western Blot analysis for each respective cell line alongside pre-stained peqGold protein marker-V (VWR, Erlangen, Germany). Proteins were separated according to size using a Laemmli buffer system and 8% polyacrylamide separation gel. Two ARV7 antibodies, mouse-AG10008 (Precision, Columbia, MD, USA; 2 µg/mL) and rabbit-EPR15656 (Abcam, Cambridge, United Kingdom; 1.5 µg/mL) were applied in dilutions according to the supplier’s instruction manual in 5% milk powder. Alpha-tubulin was used as a loading control (Cell Signaling Technology, Danvers, MA, USA). Species specific secondary antibodies (horseradish peroxidase conjugated, DAKO, Glostrup, Germany) were applied at 1:10.000 dilution in 5% milk powder. Protein bands were visualized using SignalFire™Plus ECL reagent (Cell Signaling Technology, Danvers, MA, USA) and X-ray films (CEA, Hamburg, Germany) according to the instruction manual.