Isolation and Analysis of Rab37-Specific Vesicles

Vesicle isolation protocol was as described in our previous study 23 (link). Jurkat (2 × 10⁶), THP1 (1 × 10⁷) or RAW264.7 cells (1 × 10⁷) were sonicated and subjected for vesicle enrichment by two-speed of centrifugations (3,000 g for 10 min followed by 30,000 g for 60 min at 4 °C) using a 40-Ti rotor (Beckman). The vesicles-containing solution (500 μg) was incubated with anti-V5-tag antibody to isolate Rab37-specific vesicles and the CHI3L1 cargos in vesicles were analyzed by immunoblotting (Table S2).

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Yang P.S., Yu M.H., Hou Y.C., Chang C.P., Lin S.C., Kuo I.Y., Su P.C., Cheng H.C., Su W.C., Shan Y.S, & Wang Y.C. (2022). Targeting protumor factor chitinase-3-like-1 secreted by Rab37 vesicles for cancer immunotherapy. Theranostics, 12(1), 340-361.

Publication 2022

40-Ti rotor

Anti antibody Centrifugations Isolation Raw264 7 cells

Corresponding Organization : National Cheng Kung University Hospital

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Variable analysis

independent variables

Cell type (Jurkat, THP1, or RAW264.7 cells)

dependent variables

Presence and levels of CHI3L1 cargo proteins in Rab37-specific vesicles

control variables

Centrifugation conditions (3,000 g for 10 min, followed by 30,000 g for 60 min at 4 °C)
Rotor used (40-Ti rotor from Beckman)
Antibody used for isolation (anti-V5-tag antibody)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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