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. Author manuscript; available in PMC: 2022 Mar 15.
Published in final edited form as: J Immunol. 2021 Feb 10;206(6):1329–1336. doi: 10.4049/jimmunol.2000969

Figure 4. Phosphotyrosine signal is reduced in released ASC dimers and trimers from ABL KO THP-1 cells.

Figure 4.

ASC oligomerization and phosphorylation were induced by LPS+ATP stimulation (30 min+30 min) of THP-1 and ABL KO cells. Oligomers from cell culture media were stabilized by DSS crosslinking and detected by polyclonal ASC antibody while phosphorylation was detected with anti-phosphotyrosine 4G10 antibody using ECL (A) or Li-COR (B) detection systems. After 4G10 antibody ECL detection, the membrane was stripped and re-blotted with polyclonal ASC antibody. With LI-COR detection system the monoclonal 4G10 and polyclonal ASC antibodies were probed together using two secondary antibodies, one anti-mouse conjugated with red tag (for 4G10) and the other anti-rabbit conjugated with green tag (for ASC). ASC monomer (22 kDa), and dimer (44 kDa) are shown with arrows and oligomers (66 kDa and up) denoted by line. Densitometry data are expressed as a ratio of intensity of P-tyrosine bands to the corresponding ASC bands. Two immunoblots are presented out of 4 experiments performed.