P100 52

Western blotting was performed as previously described (27 (link)). Briefly, proteins were extracted using RIPA buffer (Gensharebio, Xi’an, China). Protein concentration was measured with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal quality of protein was loaded in 10% SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with primary antibodies. The primary antibodies employed were Bcl-6 (BioLegend), CXCR5 (EMD Millipore, Billerica, MA, USA), IL-21 (Abcam, Cambridge, UK), STAT3, Phospho-STAT3, iKK-α, Phospho-iKKα/β, NF-κB-inducing kinase (NIK), P100/52, and B lymphocyte-induced maturation protein-1 (Blimp-1) (all from Cell Signaling Technology, Danvers, MA, USA).

Primary CLL cells were cultured for 24 h on 3T3 or 3T40 fibroblasts, supplemented with IL‐21 or IL‐4. Subsequently, the cells were fixed, permeabilized, and attached to poly‐d‐lysine‐coated glass slides. The following primary antibodies were used: STAT3, STAT6, p‐p65, p65, and p100/52 (Cell Signaling). isPLA was performed using anti‐mouse PLUS and anti‐rabbit MINUS probes, according to the manufacturer's instructions (Merck, Darmstadt, Germany). Slides were analysed by confocal microscopy and quantification was performed using leica analysis software (Leica, Wetzlar, Germany).