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2 protocols using low fluorescence pvdf transfer membranes

1

Investigating C5a-Mediated Neuronal Signaling

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Cell culture materials and reagents were from Corning (Manassas, VA, USA). Recombinant C5a and TrkA specific antagonist (AG-879) were from R&D Systems (Minneapolis, MN, USA). Anti-ß III tubulin and anti-Fibroblast Surface Protein (FSP) were from Abcam (Cambridge, UK). Anti-C5aR and anti-β-actin antibodies were respectively from Proteintech (Chicago, IL, USA) and BioLegend (San Diego, CA, USA). Fluorescent secondary antibodies were from Life Technologies (Grand Island, NY, USA), HRP-conjugated secondary antibody was from KPL (Gaithersburg, MD, USA) and IRDye secondary antibodies were from LI-COR (Lincoln, NE, USA). Low-fluorescence PVDF transfer membranes were from Thermo Scientific (Waltham, MA, USA). The C5aR antagonist (W54011), and chambers to evaluate the neurite outgrowth (AXIS) were from EMD Millipore (Darmstadt, Germany). The human beta NGF ELISA kit and lipoteichoic acid (LTA) were from Sigma-Aldrich (St. Louis, MO, USA), and chemicals were from Fisher Chemical (Nazareth, PA, USA). Human neurons, neuronal medium and neuronal growth supplement were from Sciencell (Carlsbad, CA, USA). All experimental protocols used for this study were in accordance with the guidelines according to the Institutional Animal Care and Use Policy and approved by the IRB Protocol Committee at the University of Illinois at Chicago.
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2

Protein Extraction and Western Blot Analysis

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Adherent cells were detached with trypsin (0.4% w/v solution, Gibco) and lysed on ice with RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), 1× Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). After incubating on ice for 40 min, the samples were centrifuged for 20 min at 15 000g, 4°C. Protein concentration of the supernatants was determined using the DC protein assay kit (Bio-Rad). Protein samples were prepared in 1× Laemmli loading buffer (Bio-Rad) with DTT and resolved on 7%, 8% or 12% Mini-PROTEAN TGX™ Precast Gels (Bio-Rad), using Tris/Glycine/SDS running buffer (Bio-Rad). After electrophoresis, proteins were transferred to low-fluorescence PVDF transfer membranes (Thermo Scientific) and blocked with 3% bovine serum albumin (BSA) in PBS, 0.1% Tween for 1 h at room temperature. Membranes were incubated overnight with primary antibodies at 4°C (Supplementary Table 2) in PBS with 3% BSA and 0.1% Tween, at 4°C and, after washing, incubated with the appropriate secondary antibody (Supplementary Table 2) for 1 h at room temperature. Proteins were detected using SuperSignalTM West Pico PLUS chemiluminiscent substrate (Thermo Scientific) and immunoblots were acquired via an iBright FL1500 Imaging System and quantified with iBright Analysis Software.
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