Anti lc3b antibody

MEFs were washed three times with PBS and then incubated for 10 min in cold lysis buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100 and a protease-inhibitor cocktail (Roche)). After 10 min of rotation on a wheel, cell lysates were centrifuged for 15 min at 13,000 RPM at 4°C to sediment cell debris. Protein concentration of these clear lysates was determined using the BCA (Bicinchoninic acid) protein assay (Pierce). Fifteen micrograms of proteins were separated by SDS-PAGE 12% and then, transferred onto polyvinyl difluoride (PVDF) membranes. Membranes were blocked for 1 h in PBS containing 0.1% Tween 20 and 2% of blocking agent (GE Healthcare), then incubated for 2 h with a primary monoclonal anti-LC3B antibody (NanoTools, Germany) and a secondary anti-mouse antibody conjugated to horseradish peroxidase (HRP). The activity of HRP was revealed by enhanced chemiluminescence (Perkin-Elmer).

For immunofluorescence, monocytes from CGD patients or controls were grown in supplemented RPMI and placed on microscope glass slides at 37°C for adhesion. Slides were then washed with PBS and fixed with 4% of paraformaldehyde. Cells were incubated in blocking solution (PBS-3% BSA-0.1% Triton X-100) with anti-LC3B antibody (Nanotools) and anti-HIF-1α (Abcam). After overnight staining with primary antibodies, slides were washed and incubated with anti-IgG and rabbit-TRITC (Sigma-Aldrich). Alexa Fluor 488 phalloidin was used for selective labelling of F-actin. LC3B (Abcam), Tβ4 (ABclonal), HIF-1α, and NLRP3 (Abcam) staining of lung sections were performed as described. Nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope (BX51; Olympus) and analySIS image processing software (Olympus).

p62 IF staining was performed on 5-μm paraffin liver sections after rehydration, and antigen retrieval was performed by heating in 10 mM sodium citrate, 0.05% Tween-20 at pH 6.2, aldehyde quenching in 100 mM glycine and blocking in 1% BSA, 0.2% fish-skin gelatin, 0.2% Triton X-100 0.05% Tween-20 in PBS. Incubation with anti-p62 antibody (Progen, Heidelberg, Germany) was done overnight at 4 °C, followed by incubation with anti-guinea pig secondary antibody coupled to Alexa-594 (Molecular Probes, Eugene, OR, USA). The sections were mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA).
LC3B IF staining was performed on primary hepatocytes cultured on coverslips and fixed in 4% paraformaldehyde. Reactive aldehydes were then quenched with 50 mM NH₄Cl and the cells were permeabilized with 0.1% Triton-X100 and blocked in 0.2% fish-skin gelatin diluted in PBS. Incubation with anti-LC3B antibody (Nanotools, Munich, Germany) was performed for 25 min at RT, followed by incubation with anti-mouse secondary antibody coupled to Alexa-488 (Molecular Probes), and the sections were mounted in Vectashield containing DAPI.