Mmp 12

The Nuclear Extract Kit (Active Motif) was used to fractionate the cytosolic and nuclear proteins, fractions were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham) by wet blotting. Probing was performed using hNCOR2 (Abcam), β-Tubulin (Cell Signaling) and Lamin A/C (Active Motif) antibodies. For MMP12 protein detection, the cytosolic whole protein fractions (Dignam extraction) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham) by semi-dry blotting. Probing was performed by using MMP-12 (R&D) and β-actin antibodies. Signal detection and analysis was performed on the LI-COR Odyssey system. Cell compartment separation efficiency was validated by enrichment of cytosolic proteins, such as β-Tubulin or nuclear proteins such as Lamin A/C. Signal expression values of hNCOR2 and MMP-12 were calculated in semiquantitative relation to the signal expression values of β-Tubulin, Lamin A/C and β-actin following the equation target/reference.

Recombinant active MMP7, MMP12, pepsin A, or plasmin (R&D Systems) was titrated against recombinant pro-MMP9 in MMP9 reaction buffer (150 mM NaCl, 10 mM Hepes pH 7.5, 0.05% brij-035, and 5 mM CaCl₂). Reactions were analyzed by Western blots simultaneously probed with anti-total MMP9 (catalog [Cat] #819701; BioLegend, San Diego, CA, USA) and anti-F107-MMP9, and visualized with fluorescently labeled anti-mouse (LI-COR Biosciences, Lincoln, NE, USA) and anti-rabbit (LI-COR Biosciences) secondary antibodies. The SeeBlue Plus2 molecular weight marker was used for all Western blots (LC5925, Thermo Fisher Scientific, Carlsbad, CA, USA).

Mice were sacrificed after perfusion and tissues were dehydrated and embedded in paraffin. Microtomy was then performed and tissues were stained with antibodies as previously described.²² Briefly, tissue sections were processed for staining with the following antibodies against p65‐NF‐KB (1:50) (cat. no. sc‐33020; Santa Cruz), ICAM‐1 (1:50) (cat. no. sc‐7891; Santa Cruz), and VCAM‐1 (1:100) (cat. no. Ab134047; Abcam), α‐smooth muscle actin (α‐SMA) (1:25) (cat. no. MAB1420; R & D Systems), matrix metalloprotease (MMP)‐12 (1:50) (cat. no. sc‐30072; Santa Creuz), MMP‐9 (1:50) (cat. no. sc‐393859; Santa Crueuz). Following incubation in primary antibody overnight, sections were incubated with Alexa Fluor conjugated secondary antibodies (1:300) (cat. no. A21206, cat. no. A10037; Invitrogen). Counterstaining with DAPI was performed. Images were captured in TCSSP8 Lightning Confocal Microscope, Leica Microsystems.

Protein levels of a selection of putative drug target genes in ACP were examined by Western blot analysis to validate the results of microarray analysis. Snap frozen tumor samples were homogenized in RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitors (Roche). This study utilized 6 ACP samples and 3 each of AT/RT, EPN, GBM, MED, PA and normal brain (obtained from autopsy material). Proteins samples (30 μg) were resolved on a 26 well Criterion Gel (BioRad) and transferred to Immobilon PVDF membrane (Millipore). Membranes were probed with antibodies to SHH (Millipore #06-1106; 1:1000), MMP9 (Cell Signaling #3852; 1:1000), MMP12 (R&D Systems #AF917; 1:1000) and Actin (Cell Signaling #12262; 1:10,000).

Tissue homogenates were prepared as previously described.²¹ Briefly, frozen nasal tissues were weighed and homogenized with an automated homogenizer (TissueLyser LT; Qiagen) for 2 min. The homogenates were then dissolved in 0.9% NaCl (1 ml of 0.9% NaCl per 0.1 g of tissue) with 1% protease inhibitor cocktail (Sigma‐Aldrich, St Louis, Mo) and centrifuged to collect the supernatants.
The prepared tissue homogenates were assayed for tenascin C (US Biological), periostin, MMP‐2, MMP‐3, MMP‐7, MMP‐8, MMP‐9, MMP‐12, MMP‐13 and TIMP‐1, TIMP‐2, TIMP‐3, and TIMP‐4 (R & D Systems) by commercially available kits.

Where possible, the capacity of rSmCI-1 to cleave structural molecules was assessed using commercially available kits. Due to the variability of activity between buffers, we elected to use the KSRB from the Generic MMP Activity Kit for these assays. All MMPs were activated via incubation with 1mM APMA as previously described. Gelatin and collagen type 4 cleavage were examined using an EnzChek Gelatinase/Collagenase Assay Kit (Thermo Fisher Scientific), with MMP-2 (AnaSpec) and Clostridium histolyticum collagenase (Thermo Fisher Scientific) used as positive controls. Proteases were incubated with 100 μg/ml fluorescein labeled substrate in a clear-bottom black-welled 96 well plate and 495nm/515nm fluorescence readings were obtained after 20 hours incubations at 37°C using a SpectraMax M2 microplate reader (Molecular Devices). Differences between treatments were assessed via One-Way Anova.
Elastin cleavage was examined using a SensoLyte Green Elastase Assay Kit (AnaSpec). Porcine elastase (AnaSpec) and MMP-12 (R&D Systems) were used as positive controls. Cleavage of the 5-FAM/QXL 520 labeled elastin was measured in a clear-bottom black-welled 96 well plate and 490nm/520nm fluorescence readings were obtained after 1 hour at 37°C using a SpectraMax M2 microplate reader (Molecular Devices). Differences between treatments were assessed via One-Way Anova (Prism 9; GraphPad Software).