Dusp10

PBECs were lysed in buffer containing 1% Triton X and boiled for 5 min in SDS-PAGE buffer. Lysates were subjected to SDS-PAGE, and proteins were transferred to a nitrocellulose membrane. Membranes were blotted with antibodies to DUSP10 (Abcam), phosphorylated p38 (Promega), phosphorylated JNK (Cell Signaling), and actin (Sigma-Aldrich). Antibodies were detected using a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Dako). Densitometric analysis was performed using ImageJ software (version 1.50i; NIH).

Immunohistochemical staining was performed to detect the expression of CD10 (Abcam; 1:500), BCL6 (Abcam; 1:250), MUM1 (Abcam; 1:250), TP53 (Abcam; 1:500), MYC (Santa Cruz; 1:200), BCL2 (Abcam; 1:500), and DUSP10 (Abcam; 1:500) in the DLBCL cohort (n = 106). This method has been described in our previous study [28 (link)], and primary and secondary antibodies used are listed in Supplementary Table 3.

Total protein was extracted by lysing cells in NP40 buffer containing 1× protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 2 (Sigma). Nuclear fractions were prepared using hypotonic buffer according to a previously described protocol [33 (link)]. Immunoblotting was carried out with a standard protocol with primary antibodies including histone H3 (Abcam; 1:2,000), DUSP10 (Abcam; 1:1,000), γH2AX (Ser139; Abcam; 1:2,000), JNK (CST; 1:1,000), phospho-JNK (CST; 1:1,000), p38 (CST; 1:1,000), phospho-p38 (CST; 1:1,000), p53 (Abcam; 1:1,000), phospho-p53 (Abcam; 1:1,000), Bcl2 (Abcam; 1:2,000), ATF2 (Santa Cruz; 1:1,000), phospho-ATF2 (Santa Cruz; 1:1,000), and MYC (Santa Cruz; 1:1,000) (Supplementary Table 3). The protein bands were visualized using the enhanced chemiluminescence system (Millipore) and imaged with the Odyssey Infrared Imaging System (Li-Cor Biosciences).