The SFK HCK activates SYK in MYD88 mutated lymphomas cells. (A) Expression of p-LYN^Tyr396 levels by western blot analysis in MYD88^L265P BCWM.1, MWCL-1 WM cells, TMD8, HBL-1, OCI-Ly3; MYD88^Ser222Arg SU-DHL-2 ABC DLBCL cells; and MYD88^WT OCI-Ly7, OCI-Ly19 GCB DLBCL cells, Ramos Burkitt lymphoma cells, RPMI-8226, and MM.1S multiple myeloma cells. The expression levels of total LYN in these cells as well as protein loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also shown. (B) p-LYN^Tyr396 levels by western blot analysis in CD19-selected bone marrow LPC from 6 MYD88^Leu265Pro patients with WM of whom WM1 was also CXCR4^Mut and WM2-6 was CXCR4^WT, and CD19-selected peripheral blood (PB) B cells from 6 healthy donors; lysates from OCI-Ly7 GCB DLBCL cells were used for p-LYN and protein loading control. The expression of total LYN is also shown. (C) Expression of p-SYK^{Tyr525/Tyr526} levels by western blot analysis in vector-only, HCK^WT or HCK^Thr333Met transduced BCWM.1, MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Expression levels of total HCK, SYK in these cells as well as GAPDH for protein loading control are shown. (D) Changes in p-SYK^Y525/526 and p-ERK1/2^{Thr202/Tyr204} levels following HCK knockdown with doxycycline inducible shRNA1 and shRNA2 or scrambled control vector in BCWM.1 WM and TMD8 ABC DLBCL cells. p-SYK^{Tyr525/Tyr526} and p-ERK1/2^{Thr202/Tyr204} levels were detected at day 9 following 1.0 µg/mL doxycycline induction. p-ERK1/2^{Thr202/Tyr204}, a known downstream signaling component of HCK, served as a positive control for these experiments.⁴  Expression levels of total HCK, SYK, and ERK1/2 as well as GAPDH for protein loading control are also shown. (E) p-SYK^Y525/526 protein levels by western blot analysis following a co-IP with HCK protein in MYD88^Mut BCWM.1 and TMD8 cells, and MYD88^WT Ramos cell lysates. Magnetic beads only and rabbit IgG were used as co-IP experimental controls. HCK total protein was also shown as an indication of the co-IP efficiency in these cells. IgG heavy chain was shown as an indication of the quantity of antibodies used in the co-IP experiments. Above experiments were performed at least twice with representative results shown. BM, bone marrow.