Af591

Western blotting was performed, as previously described.24 (link), 25 (link) Briefly, BMDMs were lysed in 0.1% Nonidet P40 containing a protease inhibitor cocktail.²⁵ (link) Lysates were separated on a 12% Tris-HEPES Precise gel (Thermo Fisher Scientific) and transferred electrophoretically onto polyvinylidene difluoride membranes (Merck Millipore, Burlington, Mass). Membranes were blocked with 5% nonfat milk (Marvel) in Tris-buffered saline/0.1% Tween-20 before incubation with primary antibodies directed against Mer (1:000; AF591; R&D Systems, Minneapolis, Minn) and β-actin (1:50,000; A1978, Sigma). This was followed by horseradish peroxidase–conjugated secondary antibodies (1:2500; Dako, Glostrup, Denmark) and incubation with ECL prime (GE Healthcare, Fairfield, Conn). Blots were exposed to light-sensitive film (MOL7016; Scientific Laboratory Supplies, Nottingham, United Kingdom) and processed through an X-ray developer (Ecomax Processor; Photo Imaging Systems, Germany).

Mice were injected IP with α-Axl (Antigen Affinity-purified Polyclonal Goat IgG (AF854); R&D Systems, Minneapolis, MN), α-Mer (AF591; R&D Systems), control IgG (AB-108-C: R&D Systems), or 1× PBS. The α-Axl and α-Mer antibodies are known to activate their respective receptors [28 (link)]. Dosing were as follows: (1) 10 μg every other day for a total of 40 μg starting at 8 dpm (preclinical), (2) 10 μg every other day for a total of 40 μg starting at 12 dpm, (3) 40 μg at day 8 dpm, and (4) 40 μg every other day for a total of 80 μg starting at 8 dpm. We observed the greatest effect with dose 3, 40 μg at day 8 dpm, and that dose was selected for all subsequent experiments. Mice were sacrificed at varying time points post-MOG immunization, and tissues were analyzed fresh, snap frozen, or fixed in 4% paraformaldehyde (PFA) for further analysis.

Equal amounts of protein (30 μg) were loaded onto 10% (W/V) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoresed. The proteins were transferred onto PVDF membrane (Millipore) and incubated with the primary antibodies of MEGF10 (1:700, A10508, ABclonal, MA), MERTK (1:700, AF591, R&D, MN), SYP (1:800, ab52636, Abcam), Homer-1 (1:800, ab184955, Abcam) and β-actin (1:1000, MA5–15739, Invitrogen) at 4 °C overnight. The membrane was washed in TBST buffer and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (1:5000, Invitrogen) for 1 hr at RT, and then reacted with an enhanced chemiluminescence substrate (Meilunbio, Shanghai, China). The result of chemiluminescence was recorded and semi-quantified using the ImageJ software (NIH, Bethesda, MD). Bright-field image and chemiluminescent blots are merged using Tanon GIS software (www.Bio-tanon.com.cn).

Antibodies used were Mer for WB (AF591; R&D Systems), Mer for IHC (DS5MMER; eBioscience), Axl for IHC (AF854; R&D Systems), Axl for immunoprecipitation (M-20; Santa Cruz), GAPDH (MAB374, clone 6C5; Millipore), phospho-tyrosine (clone 4G10; Millipore), Gas6 (AF986; R&D Systems), CD31 (ab28364; Abcam), MARCO (MCA1849; AbD Serotec), Laminin (L9393; Sigma-Aldrich), Desmin (ab32362; Abcam), cleaved Caspase 3 (Asp175; Cell Signaling), F4/80 (MCA497; AbD Serotec), and Ki67 (65241; BioLegend). Axl, Mer, and Gas6 antibody specificity for immunohistochemistry, immunocytochemistry, immunoprecipitation, and immunoblotting was tested using samples from corresponding mouse mutants (e.g., Fig 1G and H). Secondary antibodies used for immunoblot analysis were horseradish-peroxidase-conjugated anti-goat (705-035-003) from Jackson ImmunoResearch and antimouse (NA931V) and antirabbit (NA934V) from GE Healthcare. Secondary antibodies for immunocyto- and immunohistochemistry were fluorophore-conjugated antigoat (A-11055 from Life Technologies, or 705-166-147 from Jackson ImmunoResearch), antirabbit (A-10040 or A-21206 from Life Technologies), antirat (712-545-153 or 712-165-153 from Jackson ImmunoResearch), and antimouse (A-11029 from Life Technologies, or 715-166-150 from Jackson ImmunoResearch).

Mertk ELISA was conducted on supernatants of BMDMs or sections of atherosclerotic plaques lysed in protein extraction buffer (300 mM Tris-HCl pH 8.0 and 2% SDS) (Kawashima et al., 2014 (link)). Anti-Mertk antibody (AF591, R&D Systems) was used as the capture antibody. 200 µL of culture supernatant or 10 µg equivalent of protein from plaque lysates were loaded per well. Biotinylated anti-Mertk antibody (BAF591, R&D Systems) was used as a detection antibody followed by incubation with streptavidin-HRP and colorimetric development using TMB substrate. A450_nm was used to estimate the quantity of soluble Mertk.