Monoclonal Anti-Laminin antibody produced in mouse

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	-Mouse (1:200)

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-Mouse (1:200)

Publication protocol

At the conclusion of each experiment for matricellular and ECM protein detection, conditioned media (CM) from TM cell cultures was harvested and centrifuged at 2300g for 10 minutes at 4°C. The supernatant was then concentrated (Amicon Ultra-4 Filter Unit, 10 kDa; Millipore, Milford, MA, USA), and protein content quantified using the DC Protein Assay kit adhering to manufacturer's protocols (Bio-Rad, Hercules, CA, USA). For AMPK protein detection, cells were lysed for 3 minutes on ice with cold 1× radioimmunoprecipitation buffer containing 0.5% Aprotinin, 0.1% EDTA, 1% EGTA, 0.5% phenylmethylsulfonyl fluoride, and 0.01% Leupeptin. Samples were then centrifuged at 18,000g for 15 minutes at 4°C and protein content quantified. In all experiments, equal amounts of protein were treated with 6× reducing buffer and boiled for 5 minutes. Samples were then electrophoresed in 10% SDS-PAGE, alongside a prestained protein marker (Cell Signaling Technology, Inc., Danvers, MA, USA). For CM loading control, the resultant gels were stained with 0.1% Coomassie Brilliant Blue G-250 (Bio-Rad) for 3 hours and were destained with fixing/destaining solution until clear bands were visible and contrasted well with the true blue background. Otherwise, proteins were transferred to nitrocellulose membranes (0.45-μm pore size; Invitrogen, Carlsbad, CA, USA). Membranes were blocked for 1 hour at room temperature (RT) in a 1:1 mixture of 1× TBS-T (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween-20) and blocking buffer (Rockland, Inc., Gilbertsville, PA, USA), followed by overnight incubation at 4°C with the indicated primary antibody at 1:10,000 for SPARC (Hematologic Technologies, Inc., Essex Junction, VT, USA), 1:1000 for TSP-1 (AF3074; R&D Systems, Inc., Minneapolis, MN, USA), 1:1000 for COL1 (600-401-103-0.5; Rockland, Inc., Gilbertsville, PA, USA), 1:1000 for COL4 (600-401-106-0.5; Rockland, Inc.), 1:200 for Laminin (L8271; Sigma-Aldrich), and 1:000 for p(Thr172)-AMPKα, AMPKα, AMPKα1, AMPKα2, p-ACC, and ACC (Cell Signaling). A 1:200 dilution was used for p(Ser188)-RhoA and for total RhoA (Santa Cruz Biotechnology, Dallas, TX, USA), and a 1:1000 dilution was used for Myc-Tag and for β-actin (Cell Signaling). Following incubation with primary antibody, the membranes were washed three times with 1× TBS-T and incubated for 1 hour at RT with dye-conjugated affinity purified 680 anti-mouse or 800 anti-rabbit immunoglobulin G (IgG) antibodies, respectively (IRDye; 1:10,000 dilution; Rockland, Inc.). The membranes were then washed three times with 1xTBS-T, scanned, and integrated band intensities were calculated using an infrared imaging system (Odyssey; Li-Cor, Lincoln, NE, USA).

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