Sc 24

Equivalent amounts of total protein from extracellular vesicles and cells were separated using SDS–PAGE and transferred to a polyvinylidene fluoride membrane (88520, Thermo Fisher Scientific, Waltham, MA, USA). The membrane was blocked with 5% non-fat milk in 1X TBS-T (0.05%) for 2 h at room temperature and subsequently incubated with primary antibodies anti-HSP90 α/β (1:3000, sc-13119, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HSP70 (1:1000, sc-24, Santa Cruz Biotechnology), anti-CD9 (1:250, sc-13118, Santa Cruz Biotechnology), anti-Calnexin (1:400, sc-23954, Santa Cruz Biotechnology), anti-vitronectin (1:500, sc-74484, Santa Cruz Biotechnology), and anti-β-catenin (1:500, ab2365, Abcam, Cambridge, UK) overnight at 4 °C. The membranes were washed with TBS-T and incubated with a secondary antibody (1:5000, 115-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA, or 1:3000, 65-6120, Invitrogen, Waltham, MA, USA) for 2 h at room temperature. The membranes were washed with TBS-T three times and scanned on a C-DiGit Blot scanner (LI-COR Biosciences, Lincoln, NE, USA) using an Immobilon Crescendo Western HRP Substrate (WBLUR0100, Millipore, Burlington, MA, USA). Image Studio Digits v.5.2 software (LI-COR Biosciences, Nebraska, USA) was used for image acquisition.

Western blots were performed with the following antibodies: TONSL (1:1000, HPA046494, Sigma), CAF1A/p150 (1:1000, (Quivy et al., 2004 (link))), MCM2 (1:1000, A300-122A, Bethyl Laboratories), CAF1B/p60 (1:1000, (Quivy et al., 2004 (link))), DNAJC9 (1:1000, ab150394, Abcam), HA (1:3000-5000, C29F4 #3724, Cell Signaling Technology), DAXX (1:250, HPA008736, Sigma), HJURP (1:1000-2000, HPA008436, Sigma), Flag (1:3000-20000, F7425, Sigma), H3 (1:1000-, ab1791, Abcam), H4 (05-858, Millipore), Tubulin (1:10000, ab6160, Abcam), BAG2 (1:1000, ab79406, Abcam), HSC/HSP70 (1:200, sc-24, Santa Cruz Biotechnology) and HSC70 (1:500-1000, sc-7298, Santa Cruz Biotechnology). Immunofluorescence was performed with the following antibodies: Flag (1:500, F3165, Sigma), Goat Anti-Mouse-Alexa488 (1:1000, A-11029, Thermo Scientific).