Fig 1: N-linked glycosylation is required for proper BMP/SMADs signaling. (A) Left panel. Representative immunoblots of TFR2, HFE, HJV, and TMPRSS6 proteins in total cells lysate of HuH7 sh-74 and sh-CTR cells. GAPDH is the loading control. Right panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation. (** p < 0.01; Student’s t-test). (B) Left panel. Representative immunoblots of TFR2, HFE, and TMPRSS6 in HuH7 control cells treated with DMSO (vehicle) or increasing concentration of tunicamycin (0.5, 1.0, 1.5 µg/mL). GAPDH is the loading control. Right panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation. (** p < 0.01, 1.5 μg/mL tunicamycin-treated cells vs. vehicle, °° p < 0.01 of 1.0 µg/mL tunicamycin-treated cells vs. vehicle, §§ p < 0.01 0.5 ug/mL tunicamycin-treated cells vs. vehicle; * p < 0.05, 1.5 μg/mL tunicamycin-treated cells vs. vehicle; ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). (C) Upper panel. Representative immunoblots of pSMAD1/5/8 and tSMAD1/5/8 proteins in HuH7 control cells treated with DMSO (vehicle) and increasing concentration of tunicamycin (0.5, 1.0, 1.5 µg/mL). Lower panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation (* p < 0.05, 1.5 μg/mL tunicamycin-treated cells vs. vehicle, ° p < 0.05 of 1.0 µg/mL tunicamycin-treated cells vs. vehicle. ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). (D) Quantification of HAMP gene expression in sh-CTR cells treated with DMSO and tunicamycin (1.5 µg/mL). Data are means ± standard deviation of three independent experiments. (** p < 0.01; Student’s t-test).
Fig 2: Schematic models of the mechanism of iron overload in SEC23B loss-of-function hepatic cells. Left panel. At physiological conditions, SEC23B mediates the anterograde trafficking of correctly folded proteins and post-translational modification. Correctly processed proteins (TFR2, HJV, HFE) associated in the membrane complex that mediates activation of BMP/SMADs pathway. The activation of the complex results in the phosphorylation of SMAD1/5/8 that in turn mediates the activation of the transcription of the BMP target genes, HAMP, SMAD6, ID1, and ID3. Right panel. SEC23B loss-of-function determines the decreased glycosylation of membrane proteins TFR2, HFE, and HJV (light colored), which accounts for the reduced expression and stabilization of the membrane complex. Therefore, BMP6 failed to activate the pathway, and this finally causes the suppression of HAMP gene transcription.
Supplier Page from Abcam for Anti-HFE antibody [EPR6751(2)]