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. 2022 Apr 13;13:805026. doi: 10.3389/fimmu.2022.805026

Figure 1.

Figure 1

Anti-human CD154 clone 5C8 detects equine CD154 in PBMC. Representative example of Western blot experiments: Recombinant human soluble CD154 (r hu sCD40L) and equine PBMC lysates were separated by SDS PAGE. Molecular weights (MW) in kDa are annotated as indicated by pre-stained MW markers. (A) Proteins were blotted and visualized by StainFree technology (SF). Note the weak protein band of r hu sCD40L (15–25 kDa) accompanied by BSA (55–70 kDa, strong protein band). (B) The Western blots were probed with anti-human CD154 (conjugated with AF647) and detected as Cy5 fluorescence. PBMC were used ex vivo or after medium incubation (med), PMA and ionomycin (PMA/i)-, or SEB-stimulation in vitro. Arrows indicate CD154 bands (35–40 and 15–25 kDa) detected in PBMC. Occasionally, additional bands of 100 kDa were observed (dashed arrow). (C) CD154 detection (Cy5 fluorescence) was normalized to the total protein signal (SF fluorescence). The normalized detection/protein ratios are given per MW area and PBMC sample.