Fig 2: SCIMP-TLR interaction in macrophages. Primary BMMs were treated with the TLR2 ligand Pam3CSK4 (A) or the TLR4 ligand LPS (B), cell extracts were then used for immunoprecipitation with a SCIMP antibody, and immunoblotting detected endogenous TLR2 or TLR4, Lyn and a phospho-tyrosine antibody detected phospho-SCIMP. All data are representative of three experiments. C, GST-SCIMP effector-based pull-downs were performed in LPS-treated BMMs using site-specific phosphotyrosine probes (GST-Grb2-SH2, GST-Csk-SH2, and GST-SLP65-SH2) to detect SCIMP phosphorylated at sites at Y58, Y96, and Y120, respectively, shown on three separate gels. BMM lysates represent the input samples used for these pull downs immunoblotted to detect proteins of interest. BMMs, bone marrow–derived macrophages; LPS, lipopolysaccharides; TLR, Toll-like receptor.

Fig 3: Immunoblotting validation of key proteins centering on cell membrane structure. A Automated capillary Western blot validation of ANXA1, GRB2, and ANXA4. B The expression of ANXA1 (t = 14.216, df = 4, P < 0.001), GRB2 (t = 10.805, df = 4, P < 0.001), and ANXA4 (t = 10.118, df = 4, P = 0.009) was quantified according to the corresponding bands. GAPDH was used as the internal reference marker and loading control for normalization. These data were presented as mean ± SD of three independent experiments. NS > 0.05, **P < 0.01 and ***P < 0.001, compared with the corresponding control group