Timp4

Phorbal 12-myristate 13-acetate (PMA) and Ionomycin were purchased from Sigma-Aldrich. PMA was reconstituted to 10mg/ml stocks in DMSO and was further diluted to a working concentration of 40ng/μl in AIMV medium. Imiquimod, a TLR7 agonist, was purchased from LKT Laboratories (St Paul, MN) and reconstituted to 1mg/ml in DMSO. Recombinant TIMP1, TIMP2, TIMP3, and TIMP4 were purchased from R&D Systems and were reconstituted to working concentrations in AIMV medium. The protease inhibitors GM6001 and TAPI-0 were purchased from Calbiochem and reconstituted to 10mM and 1mg/ml stock, respectively, in DMSO.
The monoclonal rat anti-hCD200 antibodies 1B9 and 3G7, and the polyclonal rabbit serum against the extracellular region of CD200 (CD200v+c), were described previously [2 (link)]. A polyclonal rabbit serum against the human CD200 receptor (CD200R1) was generated by immunization of rabbits with a fusion protein containing the extracellular region of human CD200R1 with a his-tag at the N-terminal.
Antibodies against CD19 and CD62L used in FACS analyses were purchased from Biolegend. The apoptosis detection kit for staining of Annexin V and 7AAD was purchased from BD Biosciences. The Pan-Cadherin antibody, used as a plasma membrane marker for loading controls in Western blots, was purchased from Abcam.

A qualitative commercial enzyme-linked immunosorbent assay test (Multi-analyte ELISArray kit (SABiosciences - QIAGEN, Maryland, USA) has been used to simultaneously profile the level of multiple cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, IFNγ, TNF-α, GM-CSF) in cell culture supernatants.
A cutoff of twice the absorbance value (read at 450 nm) of the negative control for every cytokine (A₄₅₀) was used and the results were reported as positive (A₄₅₀ ≥ specific cutoff) or negative (A₄₅₀ < specific cutoff).
Protein concentration of detectable cytokines IL-6, IL-8, MMP-3, MMP-13 (Boster Biological Technology, Fremont, CA, USA), TIMP-3 and TIMP-4 (R&D Systems, Minneapolis, MN, USA) was then determined in cell-free supernatants using quantitative commercially available ELISA kits.

Metalloprotease inhibitors, including BB94 (batimastat), TAPI-1 and TAPI-2 (Santa Cruz Biotechnology), and TIMP1, TIMP2, TIMP3, and TIMP4 (R&D Systems), were added at the time of GPR37L1 induction (or 5 h after transfection in the case of U87 MG cells) or 24 h after induction for TX14A experiments, and cells were then incubated for 24 h before lysis. For MG132 proteasome inhibition, cells were induced with doxycycline overnight and then treated with MG132 for 6 h before harvest. Verification of TIMP activity in vitro using activated MMP-2 and the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ was performed as per the protocol provided by R&D Systems.