Irdye 800

Cells were lysed in Laemmli sample buffer; proteins were separated by SDS–PAGE (50 μg per lane, unless otherwise noted) and transferred to PVDF membranes. Membranes were blocked with Odyssey Blocking Buffer (LiCor) and probed with antibodies against caspase-8 (Cell Signaling Technologies), FLIP (NF6, obtained from Pr. Inna Lavrik), DR4 and DR5 (ProSci, Poway, CA), GFP (Abcam, Cambridge, MA), or β-Actin (Sigma, St. Louis, MO). Binding was detected via secondary antibodies conjugated with IRDye800 (Rockland Immunochemicals) or Alexa Fluor 680 (Invitrogen) using a LiCor Odyssey scanner. Protein levels were quantified using the LiCor analysis toolbox. Average pixel intensity was calculated for uniform rectangular regions framing individual bands, and the mean pixel intensity around each rectangular region of interest was used for background subtraction.

Hippocampal neurons from each of the six groups were lysed using cold RIPA lysis buffer containing PMSF and phosphatase inhibitor. Protein concentrations of GABA_B1 receptors and NF-κB p65 were measured using the Lowry protein assay kit. Briefly, protein homogenates were resolved using SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% skim milk powder for 1 h and incubated with primary antibodies (anti-GABA_B1 receptor, 1:500; anti-NF-κB p65, 1:500; Santa Cruz, Santa Cruz, CA) at 4 °C overnight, followed by incubation with anti-rabbit IgG antibody conjugated to IRDye 800 (Rockland, Limerick, PA) for 4 h at room temperature. GAPDH was used as a loading control. Relative protein levels were determined using gray values detected by a two-color infrared imaging system (LI-COR; Lincoln, NE).

SDS-stable complex formation was analyzed under non-reducing conditions. Samples were diluted in concentrated non-reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, 22% glycerol, 2% SDS and bromophenol blue) and separated by electrophoresis on a 12% polyacrylamide gel containing SDS. Proteins were transferred to PVDF membranes by Western blotting. Membranes were blocked using Odyssey blocking buffer (LI-COR), diluted 1:1 in PBS. Staining of the proteins was performed successively for apoE and Aβ, by incubation with goat anti-apoE (1:2500, overnight at 4 °C, Meridian Life Sciences, Memphis, TN) followed by donkey anti-goat Alexa-680 (1:5000, 1 h at RT, Invitrogen, Carlsbad, CA), and rabbit anti-Aβ 40-4 (1:2500, 1 h at RT, a kind gift of Dr. van Nostrand, Rhode Island University, Kingston, RI) followed by goat anti-rabbit IRDye800 (1:10000, 1 h at RT, Rockland, Pottstown, PA). Antibody solutions were prepared in Odyssey blocking buffer (LI-COR), diluted 1:1 in PBS. Between antibody incubations, membranes were washed extensively with PBST. Protein bands were visualized and band intensities were quantified using the Odyssey infrared imaging system (LI-COR).

Baculoviruses were lysed with loading buffer containing 2% 2-mercaptoethanol, boiled for 5 min. The lysates were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes, and then probed with either of the following: an anti-PfCSP mouse mAb (2A10) or an anti-hDAF mouse mAb (anti-CD55, Merck Millipore, Temecula, CA), together with an anti-VP39 rabbit Ab [14 (link)]. Blots probed with the appropriate secondary Abs conjugated to IRDye 680 and IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA) in the same membranes were visualized using an Odyssey infrared imager (LI-COR, Lincoln, NE). The molecular weight predictions were carried out on the ExPASy server, and densitometry analyses were performed using Image Studio Digits (LI-COR).