Lamp1 rabbit mab

Antibodies for LAMP1 (sc-20011, IF 1:200); LAMP2 (sc-18822, IF 1:200); Golgin97 (sc-59820, IF 1:500); GAPDH (sc-365062, WB 1:5000); Tubulin (sc-5286, WB 1:3000) were from Santa Cruz Biotechnology. The GM130 antibody (610822, IF 1:1000) was from BD Biosciences. Flag (M2, IF 1:1000; F7425, WB 1:3000) antibody was from Sigma. LAMP1 rabbit mAb (#9091, IF 1:200); GM130 rabbit mAb (#12480, IF 1:300); Rab5 rabbit mAb (#3547, IF 1:200), Phospho-STING S366 (#50907, WB 1:1000); Phospho-TBK1 Ser172 rabbit mAb (#5483, WB/IF 1:1000) were from Cell Signaling. The following antibodies were from Proteintech: STING (19851-1-AP, WB 1:1000, IF 1:1000); LC3 (14600-1-AP, IF 1:1000, WB 1:1000, reacts with LC3A, LC3B, and LC3C); TGN46 (13573-1-AP, IF 1:1000); Golgin97 (12640-1-AP, IF 1:1000). Alexa-488/594- and Pacific Blue-conjugated secondary antibodies were obtained from ThermoFisher Scientific.

Detroit562 cells were seeded on glass coverslips (diameter 12 mm) in wells of 24−well culture plates and infected with GAS for 4 h as described above. Cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X−100, blocked with 1% BSA for 1 h, and then incubated with LC3B mouse mAb (83506, Cell Signaling Technology, Danvers, Massachusetts, USA), or LAMP1 rabbit mAb (9091, Cell Signaling Technology) at 4 °C overnight. Next morning, the cells were washed with 0.1% BSA and incubated with Alexafluor 488−conjugated or Alexafluor 555−conjugated IgG (1:1000 dilution, Cell Signaling Technology). Nuclei were stained with DAPI (ZSGB−BIO, Beijing, China). Finally, cells were observed under a laser confocal microscopy (Leica TCS SP8, Wetzlar, Germany).