TF expression, activation, and the impact of PAX5 on HCK transcription in MYD88-mutated lymphoma cells. (A) Western blot studies depicting protein expression levels of PAX5, STAT3, NF-kB (NF-κB-p65), and AP-1 complex members (JunB, c-Jun, JunD) predicted by TF promoter-binding assay and PROMO analysis as HCK promoter binding TFs in MYD88-mutated WM and ABC-DLBCL cell lines (BCWM.1, MWCL-1, TMD-8, HBL-1, OCI-Ly3, and SU-DHL-2) and MYD88 wild-type B-cell lymphoma (OCI-Ly7, OCI-Ly19, Ramos) and myeloma cells (RPMI-8226, MM.1S). The HCK protein expression levels and the phosphorylation levels of mutated MYD88–directed TFs STAT3, NF-κB-p65, and AP-1 complex members (JunB, c-Jun) were also detected. GAPDH protein expression was used to demonstrate uniform protein loading. (B) The regulation of HCK transcription by PAX5 was assessed by lentiviral knockdown of PAX5 with 2 distinct shRNAs in MYD88-mutated BCWM.1 and TMD-8 cells and compared with scrambled control vector. Quantitative RT-PCR was performed after day 5 of lentiviral transduction. HCK protein levels and knockdown efficiencies for PAX5 were analyzed by western blot at the same time as the sample collection for HCK mRNA quantification. GAPDH was used for loading control. (C) The regulation of PAX5 by mutated MYD88 was assessed by lentiviral-mediated knockdown of MYD88 in MYD88-mutated BCWM.1 and TMD-8 cells using 2 distinct shRNAs and compared with scrambled control vector. Protein levels of PAX5 are shown, and GADPH served as a protein loading control. (D) Transcriptome analysis depicting PAX5 transcript levels in CD19-selected bone marrow LPCs from MYD88-mutated WM patients, and MYD88 wild-type WM patients; peripheral CD19-selected B cells and CD19- and CD27-selected memory B cells from healthy donors; and CD138-selected bone marrow plasma cells from healthy donors. ***P < .001.