Effects of phosphatase inhibitors and proteasome inhibitor MG132 on nuclear pSTAT6 in GCB-like and ABC-like DLBCL. (A) GCB-like (SUDHL6 and SUDHL4) and ABC-like OCILY10 DLBCLs were stimulated with IL-4 (100 U/mL) for the indicated times with or without sodium orthovanadate (1 mM) or MG132 (30 μM) added immediately after or immediately before IL-4, respectively. Nuclear extract protein was immunoblotted with anti-pSTAT6, anti-STAT6, and antinucleolin antibodies. These experiments were repeated twice. (B) pSTAT6 densitometry measurement of the blots shown in panel A. The results shown represent a ratio of pSTAT6 densitometry at each condition to pSTAT6 densitometry of the same cell line stimulated with IL-4 alone, normalized by nucleolin densitometry to ensure equal lane loading. (C) GCB-like (SUDHL6 and SUDHL4) and ABC-like (OCILY10 and OCILY3) DLBCL cell lines were assessed for the presence of nuclear pSTAT6 following incubation with 1 mM pervanadate alone for 30 minutes, IL-4 (100 U/mL) alone for 30 minutes, or 1 mM pervanadate for 30 minutes followed by IL-4 (100 U/mL) for additional 30 minutes. Nuclear extract protein was immunoblotted with anti-pSTAT6, anti-STAT6, and antinucleolin antibodies. No actin was detected in the nuclear extracts (not shown).