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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.

Immunohistochemistry was performed on formalin-fixed and paraffin-embedded 4-μM tissue sections. Sections were incubated with optimal concentrations of primary monoclonal antibodies, including anti-CD38, anti-CD20, anti-CD4, anti-PD-1 (R&D, AF1086, Abington, UK) and anti-Bcl-6 (Santa Cruz biotechnology, sc-858, Santa Cruz, CA, USA) for 1 hr at RT and then incubated with biotinylated goat anti-rat, goat-rabbit and biotinylated rabbit anti-mouse antibody (Zhongshan Goldenbridge Biotech, Beijing, China), respectively. The avidin-biotin-peroxidase system with 3-amino-9-ethyl-carbazole (AEC) (red color) or Vector blue (blue color) as substrates was used to perform double staining. Based on the double immunostaining, PD-1 and Bcl-6 double positive cells in liver tissue, and CXCR5 and CD4 double positive cells in the spleen were identified as Tfh cells. The absolute number of Tfh cells was independently counted by two pathologists from five representative high-power microscopic fields (400 magnification) relative to the area most involved by the inflammatory infiltrate (28 (link)).

We used electroporation to transfect BJAB cells with human BCL6 siGENOME SMARTpool reagent or CONTROL nontargeting siRNA 1 (Dharmacon, LaFayette, CO) as previously reported [16 (link)]. Whole cell extracts prepared from the transfected cells were boiled and sheared in a reducing buffer (10% glycerol, 2 gm% SDS, 1% 1M Tris-HCL [pH 6.8], 5% 2-mercaptoethanol, and 0.005gm% bromophenol blue) and used to prepare nine Western blots as described [16 (link)]. The antibodies employed were rabbit polyclonal antibodies to BCL6 (sc-858 or sc-368, Santa Cruz Biotechnology, Santa Cruz, CA), a validated affinity-isolated Prestige antibody to RUVBL1 produced in rabbit (Sigma-Aldrich Co. LLC, Saint Louis, MO, #HPA019947), and affinity-isolated actin antibody produced in rabbit (#A2066, Sigma-Aldrich). Membranes were washed, incubated with anti-rabbit IgG (Fc), alkaline phosphatase conjugate (Promega, Madison, WI), and washed again. Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega) was used to detect protein bands. Beta-actin was used to ascertain the amount of protein loaded, and relative band intensity was normalized to the intensity of β-actin expression by scanning densitometry. BCL6 and RUVBL1 protein levels in the BCL6 siRNA-transfected cells were compared with the control nontargeting siRNA-transfected cells by an unpaired t test.