Ap substrate

Proteins extracted in SDS sample buffer from H413 clone-1 cells treated with isotype control antibody or mouse monoclonal antibody to CD24 peptide (5 µg/ml) or CD24 peptide antibody (5 µg/ml) plus c-Src inhibitor saracatinib (AZD0530, 1 µM) or active recombinant IL-18 for 3 h, were separated by PAGE using gradient 5% to 12% mini-gels, transferred to nitrocellulose membranes (Bio-Rad) and blocked overnight with 3% bovine serum albumin (Sigma) in 0.1 M Tris buffered salts solution pH 7.4 (TBS). Blotted antigens were incubated with a mouse monoclonal anti-NLRP3 (1 µg/ml, Abcam), and a rabbit polyclonal anti-β-actin (0.1 µg/ml, GenTex) as a loading control in 0.05% Tween20/TBS for 2 h, washed and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit/mouse IgG, DAKO, Denmark) diluted 1:1500 in Tween20/TBS for 2 h. Bound antibody was visualized with AP substrate (Bio-Rad) after development of reactivity for proteins from control antibody, and anti-CD24 treated cultures under standardized conditions.

H413 clone-1 cells were treated with different conditions as described above. Whole cell proteins were prepared by scraping the cells in cold PBS, extracted in SDS sample buffer, separated by SDS-PAGE using 5% to 12% gradient mini-gels, transferred to nitrocellulose membranes (Bio-Rad), and blocked overnight with 3% BSA (Sigma) in 0.1 mol·L^-1 Tris buffered salts solution pH 7.4 (TBS). The nitrocellulose membranes were then incubated with rabbit polyclonal antibodies to human occludin (ab31721), JAM-A (ab106114), claudin-1 (ab15098), claudin-4 (ab53156), ZO-1 (ab59720), claudin-15 (ab215354; all 1 μg·mL⁻¹, Abcam, Cambridge, UK), and β-actin (0.1 μg·mL⁻¹, GenTex, Zeeland, MI, USA) in 0.05% Tween20/TBS for 4 h. β-actin was used as a loading control. After the incubation, the membranes were washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1 500 in Tween20/TBS for 2 h. Bound antibodies were displayed with AP substrate (Bio-Rad) after the development of reactivity for proteins.

Extracted proteins were separated re-separated by PAGE using gradient 5 to 12% minigels, transferred to 0.2-μm nitrocellulose membranes (Bio-Rad) and blocked for ≥ 2 h with 3% bovine serum albumin (Sigma) in 0.1 M Tris buffered salts solution pH 7.4 (TBS). Blotted antigens were incubated with a mouse monoclonal anti-HSP-70 (2 μg/ml, Abcam ab2897), rabbit monoclonal anti-Beclin-1(2 μg/ml, Abcam ab207612), rabbit polyclonal antiLC3β (1 μg/ml Abcam ab51520) and rabbit polyclonal anti-p62 (2 μg/ml Abcam ab91526), 0.05% Tween20/TBS for 2 h, washed and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit/mouse IgG, DAKO, Denmark) diluted 1:1500 in Tween20/TBS for 2 h. Bound antibody was visualised with AP substrate (BioRad).