Un scan it gels version 6.1

Protein lysates prepared from different tissues were fractionated by SDS-PAGE and analyzed by Western blotting as described (Ashok et al., 2018b). In short, cells scraped from different tissues were lysed in RIPA lysis buffer (50 mM Tris-HCl pH7.4, 100 mM NaCl, 1% NP-40, 0.5% deoxycholate), boiled in reducing gel-loading buffer for 5 min at 100°C, and fractionated by reducing SDS-PAGE. Fractionated proteins were transferred to a PVDF membrane and probed for specific proteins. Quantification of hepcidin specific bands was performed by densitometry using UN-SCAN-IT gels (Version 6.1) software (Silk Scientific, USA) and analyzed graphically using GraphPad Prism (Version 5.0) software (GraphPad Software Inc., USA) and provided as fold change following normalization with β-actin or GAPDH.

Samples prepared in RIPA lysis buffer (50mM Tris-Cl pH7.4, 100mM NaCl, 1% NP-40, 0.5% deoxycholate) were clarified by centrifugation, boiled in reducing gel-loading buffer, and fractionated by SDS-PAGE. Proteins transferred to PVDF membrane were probed with specific antibodies. For fractionating ⁵⁹Fe-labeled proteins, samples were homogenized in native lysis buffer (0.14M Nacl, 1.5% Triton-X-100, 0.1 M HEPES, 1mM PMSF), fractionated by native PAGE, and visualized by autoradiography. Quantification of protein bands was performed by densitometry using UN-SCAN-IT gels (version6.1) software (Silk Scientific) and represented graphically using GraphPad Prism (Version 5.0) software (GraphPad Software Inc.). Dilutions of antibodies used for immunoblotting were Ferritin (1:1000), α-syn (1:2000), β-actin (1:10,000), RPE65 (1:1000), TfR (1:2000), α-globin (1:1000), β-globin (1:1000), HRP-mouse (1:15,000), HRP-rabbit (1:15,000).