Journal: bioRxiv

Article Title: Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations

doi: 10.64898/2025.12.20.695554

Figure Lengend Snippet: Based on their conserved domain compositions, PREB, CERS2, and NAT8F3 are new candidate MBTRs ( a ) AlphaFold-predicted structures for three potential MBTRs, here showing the mouse proteins. Transmembrane domains are marked in yellow. The domains that comprise the combination with the transmembrane domain are colored blue. ( b ) Phylogenetic trees showing 10 orthologs of each candidate protein with their Pfam domain annotations (top: PREB, middle: CERS2, bottom: NAT8F3 and NAT8F7). The TLC domain includes internal transmembrane domains. The same color scheme as panel ( a ) was applied.

Article Snippet: Mouse Preb cDNA ORF clones were obtained from Sino Biological (MG5A1071-CY) and amplified using primers designed to produce constructs encoding PREB with or without the transmembrane domain (PREBΔTM) ( Supplementary Table S1 ), with the incorporation of Notl and NheI restriction sites (NEB, R3189 and R3131).

Techniques:

Journal: bioRxiv

Article Title: Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations

doi: 10.64898/2025.12.20.695554

Figure Lengend Snippet: PREB without its transmembrane domain undergoes nuclear translocation and binds to the butyrophilin locus. ( a ) Predicted structure of PREB generated by AlphaFold, highlighting the WD repeat domains, the transmembrane domain, and the site of an engineered C-terminal truncation. ( b ) Schematic of the two constructs created to assay the mouse full-length PREB protein and PREBΔTM, lacking the C-terminal transmembrane domain. The N-terminal biotinylation site is indicated. ( c ) Western blot results of two biological replicates of ES cells expressing PREB and PREBΔTM. Two bands for PREB were detected by Streptavidin-HRP, suggesting C-terminal processing of a full-length PREB when expressed in mouse ES cells. ( d ) Immunofluorescence confocal microscopy images of PREB and PREBΔTM following Dox induction. PREB localizes predominantly to a perinuclear region, forming a ring-like shape. PREBΔTM displays diffused nuclear localization. Rightmost panels show zoomed images corresponding to the boxed regions. ( e ) bioChIP-seq profiles for PREB, PREBΔTM, BirA-only Strep-pulldown, and IgG ChIP-seq control samples. PREBΔTM samples show substantially stronger DNA-binding activity than full-length PREB. ( f ) Representative locus-specific binding profiles of PREB and PREBΔTM at the Btn1a1 (butyrophilin subfamily 1 member A1) gene. PREBΔTM shows markedly higher peak intensities, consistent with enhanced nuclear localization in the absence of the transmembrane domain.

Article Snippet: Mouse Preb cDNA ORF clones were obtained from Sino Biological (MG5A1071-CY) and amplified using primers designed to produce constructs encoding PREB with or without the transmembrane domain (PREBΔTM) ( Supplementary Table S1 ), with the incorporation of Notl and NheI restriction sites (NEB, R3189 and R3131).

Techniques: Translocation Assay, Generated, Construct, Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, ChIP-sequencing, Control, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: Scube2 Modulates Coronary Vessel Formation during Cardiac Growth and Regeneration in Zebrafish

doi: 10.64898/2025.12.17.695032

Figure Lengend Snippet: (A) SCUBE2 interacts with the PDGF-B ligand. HEK-293T cells were transfected with FLAG-tagged SCUBE2 (FLAG.SCUBE2) and His-tagged PDGF-B (His.PDGF-B) individually or in combination for two days. Cell lysates were subjected to immunoprecipitation (IP) using an anti-His antibody, and the co-immunoprecipitated SCUBE2 protein was detected by Western blot (WB) analysis using an anti-FLAG antibody (upper panel). The expression of FLAG.SCUBE2 and His.PDGF-B was confirmed by anti-FLAG and anti-His WB analysis, respectively (middle and lower panels). (B) SCUBE2 is capable of binding PDGFRβ. HEK-293T cells were transfected with FLAG.SCUBE2 and HA-tagged PDGFRβ (HA.PDGFRβ) either individually or together for two days. Lysates were immunoprecipitated using an anti-FLAG antibody, and the associated PDGFRβ protein was detected by WB using an anti-HA antibody (upper panel). The expression of FLAG.SCUBE2 and HA.PDGFRβ was validated by anti-FLAG and anti-HA WB analysis, respectively (middle and lower panels). (C) SCUBE2 promotes PDGF-BB-induced PDGFRβ signaling. HEK-293T cells were co-transfected with vector control and FLAG.SCUBE2, or with HA.PDGFRβ and FLAG.SCUBE2, for two days. PDGFRβ signaling was then stimulated by the addition of recombinant PDGF-BB protein for 30 minutes at the indicated concentrations. The levels of phosphorylated PDGFRβ (p-PDGFRβ) as indication of ligand-induced signaling, total protein of PDGFRβ, and SCUBE2 were analyzed by WB using anti-p-PDGFRβ, anti-PDGFRβ, and anti-FLAG antibodies, respectively. Protein levels of p-PDGFRβ and total PDGFRβ were quantified and plotted as a line graph (D). Statistical comparison was performed between the SCUBE2 + PDGFRβ and vector + PDGFRβ groups. Data are presented as mean ± S.D. from four independent experiments. *, P < 0.05.

Article Snippet: The His-tagged PDGF-B (catalog HG10572-NH) and HA-tagged PDGFRβ (catalog HG10514-NY) expression plasmids were obtained from Sino Biological (Beijing, China).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Binding Assay, Plasmid Preparation, Control, Recombinant, Comparison