Mab5871

Western blot was performed based on the manufacturer’s specification. Briefly, isolated brain tissues (100 mg) or primary cultured cells (5 × 10⁶) were resuspended in RIPA lysis buffer (Beyotime) with PMSF. Lysed protein was quantified by BCA protein assay (BCA, Beyotime), separated by SDS-PAGE (10%) and transferred toward PVDF membranes. PVDF blots were then incubated with primary antibodies recognizing rabbit anti-TRPV1 [(1:1,000, NB100-98886, Novus), goat anti-Iba1 (1:500, NB100-1028, Novus), mouse anti-CD68/ED1 (1:1,000, ab31630, Abcam), mouse anti-TLR4 (1:1,000, sc-293072, Santa Cruz, CA, USA), mouse anti-TGF-β1 (1:1,000, MAB240-100, R&D), rat anti-TβR I (1:1,000, MAB5871, R&D), goat anti-TβR II (1:1,000, AF532, R&D), rabbit anti-Arg1 (1:1,000, 9819, CST), goat anti-Ym1 (1:1,000, AF2446, R&D), rabbit anti-GAPDH (1:1,000, ab9485, Abcam), or mouse anti-β-actin (1:100,000, 60008, proteintech)]. After incubation with the corresponding HRP-conjugated secondary antibody, immunoreactive bands were developed by enhanced chemiluminescence (ECL) detection reagent.

Membrane extracts from fibulin-6- or scr-siRNA-transfected nCF stimulated for 24 hours with 10 nM mouse TGF-β1 were prepared using membrane extraction buffer (20 mM Tris-HCl, pH 7, 4; 1% Trition; 10% Glycerin; 1 mM EDTA) containing protease and phosphatase inhibitors (complete mini, no. 11836170001 and PhosSTOP, no.04906845001, Roche Diagnostics). 1 μg anti-TGFRII (AF352, R&D) or 1 μg goat IgG (I 5000, Vector labs) was covalently crosslinked with 3 mM bis(sulfosuccinimidyl)-suberate (BS3 (link);Thermo Scientific, Darmstadt, Germany) to 50 μl protein G dynabeads (Invitrogen, Life Technologies, Darmstadt, Germany). After washing with membrane extraction buffer antibody-protein G dynabead-complexes were incubated with 100 μg membrane extracts for 1 hour at room temperature. The complexes were washed and resuspended in 1× reducing sample buffer, boiled for 5 min and analyzed by western blot using anti-TGFRI (MAB5871, R&D) for detection.

nCF previously transfected with either fibulin-6- or scr-siRNA were detached from the culture dish by accutase (Sigma, Taufkirchen, Germany) and adjusted to 5 × 10⁵ cells per ml PBS containing 0,1% BSA, 1 mM CaCl₂ and 1 mM MgCl₂. Cells were stained with TGFRI (MAB5871, 1:10, R&D) and TRGRII (AF352, 1:10, R&D). Cy2-coupled rat or goat IgGs were used as secondary antibodies respectively. Exclusion of non-viable cells was achieved by staining with 1 μg/ml propidium iodide (Sigma, Taufkirchen, Germany).
Mean fluorescence of samples was measured with Cytomics FC500 FACS (Beckham Coulter, Krefeld, Germany) and analyzed using FlowJo 10-0-8 (FlowJo, Ashland, OR, USA).