Journal: Cell reports

Article Title: Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates

doi: 10.1016/j.celrep.2021.109840

Figure Lengend Snippet: (A) Depiction of Dfm1, Der1, and Der1-Shp. Dfm1 and Der1 are ER-localized membrane proteins with six transmembrane domains. (B) Alignment of WR and GxxxG motif from H. sapiens Derlin-1 and S. cerevisiae Der1 and Dfm1. (C) Expression levels of Der1-Shp variants are measured by loading increasing amounts of lysates (15 and 30 μL) on SDS-PAGE followed by immunoblotting with α-HA. (D) In the indicated strains, degradation of Hmg2-GFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Hmg2-GFP with α-GFP. Band intensities were normalized to PGK1 loading control and quantified by ImageJ. t = 0 was taken as 100%, and data are represented as mean ± SEM from n = 3 biological replicates, ***p < 0.001, repeated-measures ANOVA. (E) Total cell lysates (T) from the indicated strains were separated into soluble cytosolic fraction (S) and pellet microsomal fraction (P) upon centrifugation at 14,000 × g . Each fraction was analyzed by SDS-PAGE and immunoblotted for Cdc48 with α-Cdc48 and Pgk1 with α-Pgk1. The graph shows the quantification of Cdc48 in the pellet fractions of the respective cells as measured from ImageJ. Data are represented as percentage of Cdc48 that is bound to pellet fraction and is shown as mean ± SEM from n = 3 biological replicates, ****p < 0.0001, one-way ANOVA.

Article Snippet: Full-length human DERLIN-1 cDNA was obtained by G-block synthesis (Eton Bioscience, Inc.) and subcloned into pcDNA3.1/Myc-His(+)A (Invitrogen) to express Derlin-1 with the myc epitope at the C terminus.

Techniques: Expressing, SDS Page, Western Blot, Centrifugation

Journal: Cell reports

Article Title: Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates

doi: 10.1016/j.celrep.2021.109840

Figure Lengend Snippet: (A) Alignment of H. sapiens Derlin-1, 2, and 3 and S. cerevisiae Dfm1. Similarly or identically conserved residues in L1 and TM2 are highlighted in red and green, respectively. (B) DERLIN-1 KO HEK293T cells were transfected with DERLIN-1 as described under . 50 μg of lysate was subjected to immunoblotting for Derlin-1 with α-Derlin-1 and GAPDH with α-GAPDH. (C) Cycloheximide-chase were performed in DERLIN-1 KO HEK293T cells co-transfected with DERLIN-1 and ΔF508-CFTR. 48 h after co-transfection, cells were treated with 100 μg/mL CHX and harvested at the indicated chase times for immunoblotting of CFTR. (D) HEK293T lysates were incubated with Ni +2 beads, and the bound proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting with specific antibodies against Derlin-1 and CFTR. (E) Protein structure clusters with the highest prevalence (~50% of simulation time) of WT Dfm1 protein (gold) and human Derlin-1 (blue). Lipid head group densities from simulations of human Derlin-1 (red) with the protein structure (purple) and overlayed with the WT Dfm1 lipids (cyan) and protein structure (orange).

Article Snippet: Full-length human DERLIN-1 cDNA was obtained by G-block synthesis (Eton Bioscience, Inc.) and subcloned into pcDNA3.1/Myc-His(+)A (Invitrogen) to express Derlin-1 with the myc epitope at the C terminus.

Techniques: Transfection, Western Blot, Cotransfection, Incubation, SDS Page