Lsp 1

Two of the identified immune-related proteins, Cyclophilin A (CypA) and Cofilin 1 (Cof1), that were included in the list of proteins contributing to the separation of the TDI-treated group and AOO-treated group in the PCA were verified via Western blotting in B lymphocytes obtained from a new, independent set of similarly treated TDI- and AOO-treated mice (n = 10/group). Briefly, proteins were loaded and separated on 4–12% Bis/Tris Midi-gels (Invitrogen, Merelbeke, Belgium) and subsequently transferred to a PVDF membrane (iBlot, Gel Transfer Stack, Invitrogen). Membranes were blocked (1–2 h, 5% blocking agent, GE Healthcare) and incubated overnight with primary antibody (LSP-1: 1/1000, goat Ab, Santa Cruz Biotechnology; CypA: 1/1000, mouse Ab, Santa Cruz Biotechnology; GAPDH, internal standard, 1/200000, mouse Ab, Dako). Following secondary Ab incubation (LSP-1: 1/50000, donkey anti-goat IgG, Santa Cruz Biotechnology; Cof1 and GAPDH: 1/100000, goat anti mouse IgG, Dako) protein bands were visualized using chemiluminescence detection (Supersignal West Dura, Thermo Scientific) on ECL hyperfilm (GE Healthcare). The protein bands were semiquantitatively evaluated by densitometry (ImageQuant TL v2009, GE Healthcare).

The IHC staining and the quantification of staining intensity was performed as previously described with the following primary antibodies respectively (LSP1, Santa Cruz, sc-53363; IBA1, abcam, ab5076; Neurophil Elastase, abcam, ab68672) [44 (link), 46 (link)].

BST-2, CD63, CD9, CD81, TSG101, LSP-1, LAMP-1, GW182, HIV-1 Reverse Transcriptase, HIV-1 p24, Actin and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Integrin β1, Ago2, TRBP-2 and Dicer antibodies were obtained from Cell Signaling Technology (Danvers, MA). MHC II antibody were obtained from Abcam (Cambridge, MA). LFA-1 antibody was obtained from R&D systems (Minneapolis, MN). Exosome-depleted FBS was obtained from System Biosciences (Palo Alto, CA).