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. 2022 Mar 22;298(5):101857. doi: 10.1016/j.jbc.2022.101857

Figure 3.

Figure 3

Syk acts downstream of Lyn for SCIMP and TLR4 phosphorylation.A, RAW246.7 cells were pretreated with the Lyn inhibitor SU6656 for 1 h before LPS stimulation. Phospho-Syk was detected by immunoblotting. Syk phosphorylation at the peak time point is presented as mean with SEM (n = 3) and was quantified by student’s t test, ∗p < 0.05. B, V5-SCIMP was immunoprecipitated using the V5 antibody from the extracts of CRISPR-mediated SCIMP-deficient, V5-SCIMP reconstituted RAW264.7 cells. Note, the same eluate was used here (left panel) to probe for SCIMP phosphorylation and in Figure 5B. Cells were pretreated for 1 h with Lyn (SU6656) or Syk (SykIV) inhibitors before addition of LPS followed by harvesting for immunoprecipitation. C, GST-TLR4-TIR protein was immobilized on GSH beads and coincubated with LPS activated macrophage lysates for pull-down. Phospho-tyrosine was detected by immunoblotting in bead eluates. Data for panels A and B are representative of three independent experiments, and panel C is representative of two experiments. LPS, lipopolysaccharide; Syk, Spleen tyrosine kinase; TLR, Toll-like receptor.