BCR signaling is intact in R406-sensitive DLBCL cell lines. (A) SYK domains and key tyrosine residues. The SYK tandem SH2 domains (black boxes), the linker region (aa 264-370), and the kinase domain (gray box) are shown. N indicates NH₂-terminal; C, C-terminal; Y, tyrosine; P, phosphorylation. Following BCR engagement, LYN induces phosphorylation of SYKTyr348 and Tyr352 in the linker region. Thereafter, SYK undergoes autophosphorylation of SYKTyr525/526 and associated activation. (B-C) BCR signaling in R406-sensitive (B) and R406-resistant (C) DLBCL cell lines. The integrity of the BCR signaling pathway was assessed by crosslinking the B-cell receptor (+ BCR) and analyzing downstream events including the initial tyrosyl phosphorylation of SYK352, the subsequent autophosphorylation of SYK525/526 and the SYK-dependent phosphorylation of the B-cell linker protein (BLNK). In the same experiments, the specificity of R406 was assessed by incubating each of the DLBCL cell lines with the compound prior to BCR crosslinking (+ R406). Untreated cells or cells stimulated with anti-IgG/IgM in the presence or absence of R406 were then lysed and lysates size-fractionated, blotted, and analyzed with phospho-specific antibodies directed against SYK352 (top panel), SYK525/526 (middle panel), and BLNK (bottom panel). Thereafter, the blots were subsequently stripped and reprobed with anti-(total) SYK or anti-(total) BLNK antibodies as indicated.