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Fig. 3 | Molecular Medicine

Fig. 3

From: Normal Human Epidermal Keratinocytes Express In Vitro Specific Molecular Forms of (Pro)Filaggrin Recognized by Rheumatoid Arthritis-Associated Antifilaggrin Autoantibodies

Fig. 3

One-dimensional immunoblotting reactivity of MAbs, RA sera, and control sera to proteins extracted with low-salt buffer or with urea from cultured keratinocytes and normal human epidermis

Proteins were extracted either with a low-salt buffer (“LS extracts”) or with high concentrations of urea (“Urea extracts”), from cultured keratinocytes (left) and from normal human epidermis (right). In each case, after SDS-PAGE, immunoblotting analysis of the extracted proteins was performed with anti-mouse IgG alone as a control (lane 1); EE21-06, a MAb to cytokeratins 1, 2, 10/11 (lane 2); AKH1 and AHF1, MAbs to (pro)filaggrin (lanes 3, 4); AHF7, a MAb to filaggrin (lane 5); anti-human IgG alone as a control (lane 6); RA sera (lanes 7–10); normal human sera (lanes 11–14). Within the LS extracts, the RA sera and the anti(pro)filaggrin MAbs detect both 40–43 kDa bands and a diffuse high-molecular-weight antigen from 70 up to 200 kDa and above in the cultured keratinocytes, while only a diffuse 40 kDa band corresponding to the neutral/acidic isoform of filaggrin is detected in normal epidermis. Within the urea extracts, the RA sera and the anti (pro) filaggrin monoclonal antibodies reveal a diffuse antigen with molecular weight >100 kDa in the cultured keratinocytes, while they all detect the urea-soluble filaggrin in normal epidermis, AKH1 and AHF1 showing an additional diffuse reactivity towards the urea-soluble precursor profilaggrin. Note that in the urea extracts, EE21-06 as well as human sera are reactive towards cytokeratins in the 50–66 kDa zone (lanes 2, 7–14); in extracts of normal epidermis, some of them are also reactive towards aggregated cytokeratins in the 66–200 kDa zone (lanes 2, 7–10, 14). Molecular weight markers are on the left of each series of immunoblots. The migration of filaggrin is indicated on the right (arrow, F).

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