Journal: Journal of inherited metabolic disease

Article Title: YME1L1 Dysfunction Associated With 3-Methylglutaconic Aciduria.

doi: 10.1002/jimd.70029

Figure Lengend Snippet: FIGURE 3 | Effects of the c.1999C>G variant on the levels and processing of YME1L1. (A) Representative western blot and densitometry for the evaluation of YME1L1 protein levels (precursor and mature forms), using an antibody against the C-terminal part of YME1L1 (YME1L1_C, Proteintech, 11 510-1-AP). Densitometry analysis is presented as a ratio to GAPDH (n = 3). (B) Detection of YME1L1 using an antibody against the N-terminal segment of the protein (YME1L1_N, ABclonal, A10402). GAPDH served as a loading control. (C, D) Western blot of cell lysates from HeLa cells transiently transfected with either wild-type YME1L1-myc-FLAG or YME1L1L667V-myc-FLAG probed with a myc (C) or YME1L1_C (D) antibody. Non-transfected HeLa cells (C, D) and patient-derived fibroblasts (D) served as controls. GAPDH was used as a loading control. The arrow indicates a truncated protein fragment of ~40 kDa detected only in patient's fibroblasts. Data are presented as mean ± SEM, ns, not statistically sig- nificant (one-way ANOVA). m: Mature, p: Precursor, u: Unspecific.

Article Snippet: The expression vector of human YME1L1 in frame with a C- terminal myc- FLAG epitope was purchased from Origene (RC201088).

Techniques: Variant Assay, Western Blot, Control, Transfection, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Hepcidin expression is associated with increased γ-secretase–mediated cleavage of neogenin in the liver

doi: 10.1016/j.jbc.2024.107927

Figure Lengend Snippet: Hjv prevents Netrin-1–induced Neo1 degradation, and Alk3 facilitates the generation of γ-secretase–cleaved Neo1-ECD/TMD in Hep3B cells. A , diagram of fNeo1, mouse Hjv with an N-terminal 3xFLAG epitope (fHjv), mouse Alk3 with an N-terminal FLAG/MYC epitope (fAlk3), and mouse Netrin-1. B , co-expression with fHjv does not inhibit α-secretase cleavage of fNeo1. At 56 h after transfection, cell surface proteins were biotinylated. The eluted cell surface proteins, ∼10% of input lysate, and a fraction of concentrated CM were subjected to SDS-PAGE and immunodetection (IB). C , co-expression with fAlk3 increases fNeo1, fNeo1-ECD/TMD, and fNeo1-ECD. Experiments were performed as described above in ( B ) except that LY450139 (10 μM) was used to inhibit γ-secretase proteolysis. Each panel was cropped from the same image. D , quantification of cell surface fNeo1 and fNeo1-ECD/TMD bands in ( C ) (panel-1, lane 7/9), as well as fNeo1-ECD bands in ( C ) (lowest panel, lane 2/4) (n = 3). E , co-expression with fAlk3 increases γ-secretase–cleaved fNeo1-ICD. Experiments were performed as described in the legend to Fig. 3 B . Each panel was cropped from the same image. Two sets of images with different exposure for anti-FLAG antibody were presented. F , quantification of fNeo1-ICD bands in ( E ) (panel-2; lane 5/6) (n = 3). G , incubation with Netrin-1 decreases fNeo1 levels in Hep3B cells. fNeo1-transfected Hep3B cells were incubated with mouse Netrin-1 at 0, 0.1, 0.25, 0.5, or 1.0 μg/ml for ∼16 h. Cell surface proteins were subjected to biotinylation and immunodetection. H , quantification of total cell surface fNeo1 in ( G ) (panel-4) (n = 3). I , co-expression of Hjv and Neo1 prevents Netrin-1–mediated degradation of Neo1. Cells were transfected as described above in ( B ) and incubated with mouse Netrin-1 at 0 and 0.5 μg/ml for ∼16 h, followed by biotinylation and immunodetection. All experiments were repeated at least three times with consistent results. All quantification data shown are means ± SD. ∗, p < 0.05; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: Mouse Neo1 ORF (NM_008684) with a C-terminal FLAG/MYC epitope (fNeo1) in pCMV6 vector (#MR226235) and mouse Alk3 ORF (NM_009758.3) with a C-terminal FLAG/MYC epitope (fAlk3) in pCMV6 vector (#MR227586) were obtained from OriGene Technologies Inc. We obtained pEGFP-N1 plasmid from Clontech.

Techniques: Expressing, Transfection, SDS Page, Immunodetection, Incubation