Lamp1 sc 19992

BMDCs were placed on glass coverslips that had been treated with poly-l-lysine at room temperature for 30 min. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 60 min. Paraformaldehyde-fixed cells were permeabilized with 0.2% Triton X-100 in PBS. After blocking with 5% goat serum, the cells were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies. The primary antibodies used were ATP6V1a (ab137574, dilution 1:100; Abcam) and LAMP1 (sc-19992, dilution 1:200; Santa Cruz Biotechnology). Cells were mounted with DAPI, and fluorescence was visualized with a Nikon C2 confocal microscope. The image was processed with Imaris 8.4 software. Mander’s colocalization coefficient was calculated using ImageJ (National Institutes of Health). Mander’s M1 was used to determine the degree of the green channel that colocalized with the red channel.

Primary antibodies used for Western blotting and immunofluorescent staining were as follows: anti–syntaxin 7 (AF5478; R&D Systems), VAMP8 (104302; Synaptic Systems, Goettingen, Germany), MPO (HM1051; Hycult Biotech, Uden, Netherlands), TLR9 (PA5-20203; Thermo Fisher Scientific, Asheville, NC), phosphorylated Erk and total Erk (9101, 9202; Cell Signaling Technology, Danvers, MA), phosphorylated P38 and total P38 (9211, 9212; Cell Signaling Technology), Flag (CGAB-DDK; Genecopoeia), GFP (A6455; Life Technologies, Carlsbad, CA), LAMP1 (sc-19992; Santa Cruz Biotechnology, Santa Cruz, CA), Myc (sc-40; Santa Cruz Biotechnology), Munc13-4 (described previously; Brzezinska et al., 2008 (link)), and anti-Rab7 (D95F2; Cell Signaling Technology).

3-Methyladenine (3-MA) (S2767), Rapamycin (S1039), and Wortmannin (S2758) were purchased from Selleckchem (Houston, TX, USA). Chloroquine diphosphate salt (C6628) was purchased from Sigma-Aldrich (MO, USA). cis-Diamminedichloroplatinum (Cisplatin, DDP, Lot.No:8L012A88) was purchased from QILU Pharmaceutical (Shandong, China). Antibodies against ATG5 (ab109490), ATG7 (ab133528), MITF (ab140606), and LC3B (ab192890) were purchased from Abcam (Cambridge, UK). Antibodies against p62 (18420-1-AP) and GAPDH (60004-1-Ig) were purchased from Proteintech (Chicago, IL, USA). Antibody against LC3B (83506S) and Beclin 1 were purchased from Cell Signaling Technology (Danvers, MA, USA). LAMP-1 (sc-19992) and LAMP-2 (sc-18822) were purchased from Santa Cruz (Dallas, TX, USA).