Fp 8000 fluorescence spectrometer

For fluorescence resonance energy transfer (FRET)‐based lipid mixing analysis, liposomes were prepared at a 100 : 100 : 4 : 1 molar ratio of DOPC, DOPS, NBD‐DOPE, and Rho‐DOPE. The FRET liposomes (100 µm lipid) and PD‐1 EVs (60 µg·mL⁻¹ protein) were mixed and incubated for 30 min at 27 °C under acidic conditions (pH 4.5) or neutral conditions (pH 7.5). In the antibody fusion inhibition test, PD‐1 EVs were pre‐incubated with anti‐gp64 antibody (sc‐65499; Santa Cruz Biotechnology) or mouse IgG2b isotype control antibody (401201; BioLegend, San Diego, CA, USA) diluted to a concentration of 0.2 or 2 µg·mL⁻¹ in phosphate‐buffered saline at room temperature for 2 h. 7‐Nitro‐2‐1,3‐benzoxadiazol‐4‐yl (NBD) fluorescence intensity (excitation at 460 nm, emission at 535 nm) was measured using a FP8000 fluorescence spectrometer (Jasco, Tokyo, Japan). NBD fluorescence recovery was calculated using:
NBD fluorescence recovery (%) = (F – F_min)/(F_max – F_min)
where F_min represents the NBD fluorescence intensity of FRET liposomes before addition of PD‐1 EVs and Fmax represents the NBD fluorescence intensity of DOPC/DOPS/NBD‐DOPE (100 : 100 : 4 molar ratio) liposomes (100 µm lipid).

Substrate binding assay was performed as described (Bösl et al., 2005 (link)). Complex formation between Hsp104 E284Q and fRCMLa or between Ssa1-Ydj1 and fRCMLa was detected by following fluorescence anisotropy signal, measured in a JASCO FP-8000 Fluorescence Spectrometer. fRCMLa (1 μM) was incubated at 30ºC in the renaturation buffer, containing 2.6 mM ATP and at the increasing concentrations of ADP. Subsequently, Hsp104 E285Q (12 μM) or Ssa1 (2 μM) with Ydj1 (0.5 μM) were added to the reaction mixture. After 200 s, fRCMLa release from Hsp104 was induced by addition of non-labeled RCMLa to the final concentration 40 μM.

fRCMLa (1.25 μM, 5 μM or 20 μM, as indicated) was incubated for 2 min with ClpP at 30 ºC and subsequently HAP was added. Reaction was carried out in the renaturation buffer, containing 2.6 mM or 10 mM ATP and, optionally, ADP or/and Ssa1 and Ydj1, as indicated. fRCMLa proteolysis by HAP-ClpP was monitored by following fluorescence anisotropy in the JASCO FP-8000 Fluorescence Spectrometer.