Hpa029292

BJAB cells were transfected with human BCL6 siGENOME SMARTpool reagent or CONTROL nontargeting siRNA 1 (Dharmacon, LaFayette, CO) by electroporation as described previously [23 (link)]. Whole cell extracts were prepared from the transfected cells and subjected to Western blotting as described. The antibodies used included rabbit polyclonal antibodies to BCL6 (sc-858 or sc-368, Santa Cruz Bio-technology, Santa Cruz, CA), a validated affinity-isolated Prestige antibody to ITM2B produced in rabbit (Sigma-Aldrich Co. LLC, Saint Louis, MO, #HPA029292), and affinity-isolated actin antibody produced in rabbit (#A2066, Sigma-Aldrich). The membranes were washed and incubated with anti-rabbit IgG (Fc), alkaline phosphatase conjugate (Promega, Madison, WI), then washed again. Protein bands were detected with Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega).
Calculations of relative band intensity on four Western blots were normalized to the intensity of β-actin expression by scanning densitometry. The paired t test was used to compare BCL6 and ITM2B protein levels, respectively, in the siRNA BCL6-transfected cells with the corresponding control cells.

Human paraffin-embedded lymphoma blocks were retrieved from the Surgical Pathology archives under an institutional review board-approved protocol. In each case, the sections that are stained with the different antibodies are from the same tissue block, but they are not necessarily consecutive sections. BCL6 staining was performed as described previously [24 (link)] with mouse monoclonal anti-human BCL6 (clone LN22; Novocastra). Staining for ITM2B was performed overnight at 4 °C with an affinity-isolated Prestige antibody produced in rabbit (Sigma-Aldrich, Saint Louis, MO, #HPA029292) that was diluted 1:20–1:25. Antigen–antibody binding was detected with DAB chromogen, and tissues were counterstained with hematoxylin. Images were taken with a BX41 microscope (Olympus), DP72 digital camera, and cellSens Standard imaging software (Olympus).