Journal: iScience

Article Title: A CHD8-TRRAP axis facilitates MYC and E2F target gene regulation in human neural stem cells

doi: 10.1016/j.isci.2025.111978

Figure Lengend Snippet:

Article Snippet: Probed blots were incubated at RT with HorseRadish Peroxidase (HRP) conjugated secondary anti-mouse (A90-116P, Bethyl) and anti-Rabbit (A120-101P, Bethyl) antibodies, respectively.

Techniques: Virus, Recombinant, Membrane, Protease Inhibitor, SYBR Green Assay, Staining, cDNA Synthesis, Mass Spectrometry, shRNA, Sequencing, Control, Plasmid Preparation, Software, Sonication

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet: ( A ) Emetine- and micrococcal nuclease-treated membranes from wild-type (WT) or 3xFlag-TMCO1 (Flag) HEK293 cells were digitonin-solubilized, immunoprecipitated via the 3xFlag tag on TMCO1, and the eluate sedimented through a sucrose cushion to isolate the ribosome-associated fraction for analysis. ( B ) Proteins enriched in the ribosomal fraction after immunoprecipitation from 3xFlag-TMCO1 or wild-type membranes. ( C ) Top hits were confirmed by western blotting. The catalytic STT3A subunit of the OST complex is not detected. ( D ) Topology and domain structure for the top hits, based on Uniprot annotation, homology modeling, de novo structure prediction (in RaptorX-Contact), and experimental mapping; the Sec61 complex is not shown. Distinguishing features include the large globular luminal domain of Nicalin (in contrast with the flexible luminal domains of NOMO and CCDC47), the large globular cytosolic domain of CCDC47 (with a conserved C-terminal coiled-coil), and a conserved cytosolic coiled-coil between the first two TMDs of TMCO1. TMEM147 is the core, multi-pass subunit of the Nicalin-TMEM147-NOMO complex ; note that the short extra-membrane loops of TMEM147 make it difficult to detect by mass spectrometry.

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: Immunoprecipitation, Western Blot, Membrane, Mass Spectrometry

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet: ( A ) Comparison of co-purifying components in the ribosome-bound fraction (“Pellet”), following immunoprecipitation from digitonin-solubilized microsomes isolated from HEK293 cells stably expressing 3xFlag-TMCO1 or 3xFlag-Nicalin. ( B ) Digitonin-solubilized microsomes from 3xFlag-TMCO1 HEK293 cells were cleared of ribosomes by sedimentation. The ribosome-free fraction (“Input”) was then subjected to affinity purification using anti-Flag or anti-NOMO antibodies. Immunoprecipitation of 3xFlag-TMCO1 recovers only trace amounts of the other components, and NOMO1 immunoprecipitation fails to recover either TMCO1 or CCDC47. However, as shown previously , Nicalin is efficiently recovered by NOMO immunoprecipitation.

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: Comparison, Immunoprecipitation, Isolation, Stable Transfection, Expressing, Sedimentation, Affinity Purification

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet: ( A ) Density for the 80S ribosome, A/P and P/E tRNAs is from the sharpened global map after low-pass filtering by local resolution. The translocon density is from the unsharpened focused map after low-pass filtering by local resolution; isolated densities for Sec61 (green), TMEM147 (purple), TMCO1 (blue) and CCDC47 (violet) are shown at a single threshold. The focused map is also shown at a lower threshold (transparent) to highlight luminal density and the micelle. ( B ) Closeup of the Sec61 complex, including experimentally observed cross-links (red) between Sec61γ and the indicated ribosomal subunits (yellow). ( C ) Closeup of the TMEM147-Nicalin complex (purple, pink), and cross-links between uL24 and the conserved TM3-TM4 loop of TMEM147. ( D ) The luminal domain of Nicalin (pink) extends below TMEM147 in a large lobe of density. ( E ) Closeup of TMCO1, and multiple intra- and inter-molecular cross-links. ( F ) Closeup of the cytosolic domain of CCDC47 and cross-links to the indicated ribosomal subunits, Sec61α, and the TMCO1 coiled coil; a cross-link that exceeds the distance cutoff of 35 Å is in black. Density in panels B-F is from the unsharpened signal-subtracted map after low-pass filtering by local resolution.

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: Isolation

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet: ( A ) Amino acid sequence of human CCDC47 colored by ConSurf conservation score. The N-terminal signal peptide (SP) and single transmembrane helix (TM) are indicated. ( B ) Heat map of the RaptorX-Contact probabilities of two residues being in close proximity (Cβ-Cβ distance <8 Å); higher probabilities are darker. ( C ) Top five scoring models from RaptorX-Contact; each model is color ramped from the N-terminus (blue) to C-terminus (red). ( D ) High-probability (p ≥ 0.80) RaptorX-Contact contacts (white lines) mapped onto the final CCDC47 model after fitting into the cryo-EM density map.

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: Sequencing, Cryo-EM Sample Prep

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet: ( A ) Closeup view showing TMCO1 (blue), CCDC47 (violet), TMEM147 (purple), Nicalin (pink) and the Sec61 complex arranged near the nascent polypeptide (orange spheres) at the mouth of the ribosome exit tunnel. ( B ) View of the translocon from the membrane (the Nicalin luminal domain was omitted for clarity). ( C ) Surface representation of the ribosome large subunit showing regions that contact Sec61, TMEM147, TMCO1 and CCDC47.

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: Membrane

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet: Orthogonal views of the ( A ) TMCO1- ( B ) OST translocons (PDB ID 6FTI) , aligned on the Sec61 complex. The primary ribosome contacts are mediated by CCDC47 (violet), TMCO1 (blue) and TMEM147 (purple) in the TMCO1 translocon, and by RPN1 (orange) in OST; these binding sites are non-overlapping. By contrast, there are extensive steric clashes between the luminal domains of Nicalin (pink) and STT3A (yellow). In addition, TMDs of TMCO1, TMEM147 and Nicalin overlap with TMDs of the STT3A (catalytic subunit), DC2 and OST4 in the OST complex. Note the presence of an unassigned helix in the OST structure, proposed to belong to the opsin substrate TMD (red), which resides near the large, lipid-filled cavity observed in the TMCO1 translocon.

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: Binding Assay

Journal: eLife

Article Title: An ER translocon for multi-pass membrane protein biogenesis

doi: 10.7554/eLife.56889

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal CCDC47 , Bethyl , Cat# A305-100A, RRID: AB_2631495 , WB (1:1000).

Techniques: CRISPR, Recombinant, Sequencing, cDNA Synthesis, SYBR Green Assay, Software, Expressing