Atg2a

ATG2A (#15011), ATG5 (#2630), ATG7 (#2631), BECN1 (#3738), BIRC2 (D5G9), CASP8 (1C12), FADD (#2782), FAS (C18C12), FASLG (#4273), HSP90 (C45G5); LAMP1 (C54H11), MLKL (D2I6N), PARP1 (46D11), RIPK1 (D94C12), RIPK3 (D4G2A), ULK1 (D9D7), and XIAP (3B6) antibodies were obtained from Cell Signaling. SQSTM1 (#ab56416) and STX17 (#ab116113) antibodies were purchased from Abcam. β-actin (ACTB; AC-74) and ATG2B (#HPA019665) were purchased from Sigma. MAP1LC3B (#NB100-2220) was purchased from Novus Biologicals. Cell lysis, co-immunoprecipitation, subcellular fractionation, and western blotting were performed as previously described^{19 (link),41 (link)}. Relative densities of the target bands to the reference bands was calculated using ImageJ (NIH).

Whole cell lysates and equal numbers of EVs (3.75 × 10⁸ EVs) were derived from unmodified 4T1 and PalmReNL‐4T1 cells and mixed with 4× sample buffer (Bio‐Rad) with β‐mercaptoethanol (for detecting TSG101, ALIX, Flotillin‐1, and anti‐RFP) or without β‐mercaptoethanol (for detecting CD63). Proteins were separated on a 4–20% Mini‐PROTEAN TGX gel (Bio‐Rad) and transferred to a polyvinylidene difluoride membrane (Millipore, IPFL00010). After blocking with 5% ECL Blocking Agent (GE Healthcare, RPN2125) at RT for 1 h, membranes were probed with primary antibodies overnight at 4 °C at dilutions recommended by the suppliers as follows: anti‐Alix (Proteintech, 12 422), TSG101 (Proteintech, 14497‐1‐AP), flottilin‐1 (BD, 610 820), anti‐RFP (Rockland Immunochemicals, 600‐401‐379), CD63 (Thermo Fisher Scientific, 10628D, Ts63), Atg2A (Cell Signaling, 15 011), Atg2B (Cell Signaling, 25 155), Atg5 (Cell Signaling, 12 994), Atg9 (Cell Signaling, 13 509), followed by incubation with horseradish peroxidase HRP conjugated secondary antibodies at RT for 1 h. The membranes were visualized with ECL select Western Blotting Detection Reagent (GE Healthcare, RPN2235) on ChemiDoc MP Imaging System (Bio‐Rad).