Ab216513

Total proteins were isolated with RIPA buffer, separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. The membrane was blocked and incubated with primary antibodies and secondary antibodies in order. Immobilon western chemilum HRP substrate (Millipore, MA, USA) was used to visualize the protein bands. Primary antibodies used were anti-GAPDH (ab181602, Abcam), anti-TLR4 (ab13867, Abcam), anti-NF-κB p65 (ab16502, Abcam), anti-NF-κB p65 (phospho S536) (ab86299, Abcam), anti-IRAK4 (ab5985, Abcam), anti-IRAK4 (phospho T345) (ab216513, Abcam), anti-IL-6R (ab128008, Abcam), anti-STAT3 (ab119352, Abcam), anti-STAT3 (phospho Y705) (ab128008, Abcam), anti-cleaved caspase-3 (ab2302, Abcam), anti-Bcl-2 (ab182858, Abcam), and anti-cleaved PARP1 (ab32561, Abcam).

Lymph node biopsies of CLL‐infiltrated and tumour‐free samples were formalin‐fixed, paraffin‐embedded, arranged in Tissue Microarrays and stained for pSTAT6 (ab28829, Abcam) and pIRAK4 (ab216513, Abcam). The slides were analysed using Qupath (Bankhead et al, 2017 (link)) and the recommended protocol.