Anti lamp1 antibody

Cells were trypsinized, resuspended in flow cytometry buffer (2% FBS in PBS) and incubated with anti-LAMP1 antibody (Cell Signaling Technology, Danvers, USA; recognizes the luminal/extracellular domain [29-382] of LAMP1) in flow cytometry buffer at 4 °C for 30 min. After washing two times with flow cytometry buffer, cells were stained with Alexa Fluor-conjugated secondary antibodies at 4 °C for 30 min. As an isotype control, cells were stained with secondary antibodies alone. After washing, 10,000 cells per sample were analyzed using a FACSAria III flow cytometer (BD Biosciences, Franklin Lakes, USA). The data were collected and analyzed using BD FACSDiva Software (BD Biosciences). The mean fluorescence intensity of LAMP1 was collected from three independent experiments and normalized against the control group.

The expression of LAMP-1 and TSG101 in exosomes, and the expression of KLF6 in M0 macrophages were measured by using western blot. RIPA lysis buffer was used to isolate total protein. Equal quality 25 µg of cellular protein from each group were separated on the 12 % SDS-PAGE gel, and then were transferred to a PVDF membrane. After that, membranes were blocked with 5 % non-fat mile for 1 h at room temperature and subsequent primary antibodies overnight at 4 °C. The primary antibodies were as follows: anti-LAMP-1 antibody (1:1000, Cell Signaling Technology, Boston, MA, USA), anti-TSG101 antibody (1:1000, Cell Signaling Technology) and anti-KLF6 antibody (1:1000, Cell Signaling Technology). After incubation with primary antibodies, the membranes were maintained with secondary antibodies (1:3000, Abcam) for 1 h at room temperature. At last, the membranes were maintained with the enhanced chemiluminescence reagent (Merck Millipore, Missouri, USA) to detect the protein bands. The relative expression level of KLF6 was analyzed utilizing Image J software (National Institutes of Health, Maryland, USA). Here, β-actin served as the internal reference of KLF6.

We seeded HUVECs on 24-well plates, fixed them with 4% PFA, and incubated them with anti-p16 antibodies (1:200, Cat# 10883-1-AP, Proteintech), anti-p21 antibody (1:200, Cat# 2947, Cell Signaling Technology, USA), anti-Lamp1 antibody (1:200, Cat# 9091, Cell Signaling Technology), and anti-p62 antibody (1:200, Cat# 88,588, Cell Signaling Technology) overnight at 4 °C and secondary antibodies (1:1000; Cat# A11012, Cat# 11,001; Invitrogen) at room temperature, with DAPI staining nuclei for 10 min, and the cells were taken by inverted fluorescence microscopy.

Vero cells were incubated with the indicated concentrations of 1519 and competitor compound in media (DMSO 1%) for 1h at 37°C in the dark. Cells were then irradiated for 7min at 365nm followed by 3min at 302nm. Cells were collected and lysed in lysis buffer (50mM HEPES pH 7.4, 150mM NaCl, 1% NP40) with protease inhibitor. Cell debris was pelleted by centrifugation at 4°C for 20min at 13,000RPM. SDS was added to the supernatants to a final concentration of 1% and the resulting mixture was subjected to click chemistry for 1h at room temperature to conjugate Alexa Fluor 488 azide (Thermo Fisher) to the 1519 alkyne (100μM TBTA, 2mM CuSO₄, 1mM TCEP, 25μM Alexa Fluor 488 azide). Proteins were precipitated 5 times with cold acetone and the pellet was solubilized in 1% SDS lysis buffer. Prior to immunoprecipitation, the SDS was diluted to <0.1% with lysis buffer with protease inhibitors added. Samples were then subjected to immunoprecipitation with a rabbit polyclonal anti-Alexa Fluor 488 antibody (1μg/sample) overnight at 4°C and eluted off the protein A/G agarose with SDS-loading buffer. Alexa Fluor 488 was imaged using a Typhoon FLA 9500 and a rabbit monoclonal anti-LAMP1 antibody (Cell Signaling Technologies). Input was stained with Coomassie dye.

The frozen RAA tissues were embedded in the optimum cutting temperature (OCT) compound and sectioned at −25°C in a cryostat. Sections (5 μm in thickness) were fixed with 4% polyformaldehyde for 15 minutes at room temperature. The sections were permeabilized with 0.1% TritonX-100 for 20 minutes followed by blocking with 10% goat serum for 1 hour at room temperature. Then, the sections were incubated with anti-Cox IV antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 200, anti-LAMP-1 antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 200, or anti-LC3B antibody (Abcam) diluted at 1 : 100 overnight at 4°C. Subsequently, the sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 500, or Alexa Fluor 594-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 500 for 1 hour at room temperature. DAPI (Solarbio Science & Technology, Beijing, China) was used to stain the cell nuclei. Finally, the fluorescence images were captured by using a laser scanning microscope and further analyzed by ImagePro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).

Vero, HEK293T and Huh7.5 cell lines were obtained from ATCC (Manassas, VA) and maintained in DMEM (Invitrogen) supplemented with 10% FBS (Gemini Bio-Products) and 0.292mg/mL L-glutamine (Invitrogen) and grown at 37°C in 5% CO₂. LAMP1 knockout and control pX459 cell lines (293T and Huh7.5) were generated by CRISPR. LAMP1 CRISPR gRNAs targeting exon 2 (5’-GAAGTTGGCCATTATGCACG-3’) and exon 6 (5’- GCTTGCTTGTCTTATGCAGA-3’) were designed using CHOPCHOP (http://chopchop.cbu.uib.no/) and cloned into a pSpCas9(BB)-2A-Puro (PX459) (a gift from Feng Zhang (Addgene plasmid #48139)) [32 (link)]. 293T and Huh7.5 cells were transfected with the CRISPR plasmids and selected with 2μg/mL puromycin. Knockout was confirmed by sequencing using primers amplifying the target site for exon 2 and exon 6 (5’-TGCACTCCAAGAGCAGCTGTCA-3’ and 5’-GACCCAGGGCACAAAAAGATGT-3’) and by immunoblot using a rabbit monoclonal anti-LAMP1 antibody (Cell Signaling Technologies) and a mouse monoclonal anti-tubulin antibody (Santa Cruz Biotechnology).