Journal: iScience

Article Title: A mechanism of target mRNA selection and activity regulation in meiosis-related RBM46-MEIOC-YTHDC2 complex

doi: 10.1016/j.isci.2026.116234

Figure Lengend Snippet: XRN2 is a new partner of MEIOC and involved in RMY-dependent RNA repression (A) Immunoprecipitated MEIOC complexes were separated by SDS-PAGE and the protein in the gel was stained with silver prior to mass spectrometry (MS). (B) Related proteins in MEIOC complex were identified by MS. (C) Cytoplasm and nuclei were separated from HEK293T cells transfected with MEIOC and YTHDC2 (M + Y). Lamin B1 was used as a nuclear marker and GADPH as a cytoplasmic marker, N = 3. (D) XRN2 protein was co-immunoprecipitated with MEIOC. IgG was used as a negative control. N = 3. (E) Western blot analysis of MEIOC and XNR2 protein. A lentivirus containing HIS-MEIOC infected HEK293T cells. The cells were lysed with RIPA buffer and then coIP assay was performed with anti-XRN2 antibody. IgG was used as a negative control. N = 3. (F) Western blot analysis of XRN2 and β-Actin from HEK293T cells transfected with XRN2 siRNAs and control siRNA, N = 3. (G) RT-qPCR analysis showing relative mRNA levels of XRN2 in HEK293T cells transfected with XRN2 siRNAs and control siRNA, N = 3. (H) Relative luciferase activities of F-Luc-Rad21 3′UTR in HEK293T cells after knockdown of XRN2 only or combined with the expression of RBM46, MEIOC and YTHDC2 (RMY). N = 3. Data are presented as mean ± SEM. p values were determined by unpaired t test with Welch’s correction (C–E) or by one-way ANOVA with Benjamini-Hochberg correction (F–H). (I) A model of RMY complex assembly and target mRNA degradation.

Article Snippet: SDS-PAGE running buffer powder , Servicebio , Cat#G2018-1L.

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Transfection, Marker, Negative Control, Western Blot, Infection, Co-Immunoprecipitation Assay, Control, Quantitative RT-PCR, Luciferase, Knockdown, Expressing

Journal: Toxins

Article Title: Cholera Toxin-Mediated Targeting of Botulinum Neurotoxin Activity to Pain-Associated Sensory Neurons

doi: 10.3390/toxins18040174

Figure Lengend Snippet: Production of ChoBot: ( a ) Schematic showing self-assembly of the AB5-linker, a CTB pentamer assembled around CTA2 fused to Linker 1, CTA2-cholera toxin A2 subunit, CTB-cholera toxin B subunits; ( b ) coomassie-stained SDS-PAGE gel showing that mixing the linker 1-AB5 and the light chain/translocation domain (LcTd) of BoNT/A attached to Linker 2 (LcTd-Linker 2) in the presence of Linker 3 (not visible due to low MW) results in formation of LcTd-Linkers-CTA2 (arrowhead). CTB5 is denoted as a pentamer of approx. 50 kDa. Note, the CTB5 and CTA2 association is not SDS-resistant and therefore only the linking of LcTd to CTA2 is visible as a slight shift in MW of LcTd-Linker 2. Boiling for 2 min in the SDS-PAGE sample buffer results in dissociation of CTB5 pentamer into monomeric B subunits. Molecular weight standards are denoted as MW; ( c ) schematic of ChoBot modelled using known X-ray structures of the BoNT/A, the SDS-resistant SNARE linking complex and cholera toxin (PDBs 3BTA, 1SFC and 1XTC, respectively).

Article Snippet: Cell culture medium was then removed and cells were lysed in 45 μL of SDS-PAGE running buffer (56 mM sodium dodecyl sulphate, 0.05 M Tris-HCl, pH 6.8, 1.6 mM EDTA, 6.25% glycerol, 0.0001% bromophenol blue, 10 mM MgCl2, 26 U/mL benzonase) by shaking at 900 rpm for 10 min. Lysed cells were then boiled at 95 °C for 3 min and then run on 12% Novex SDS–PAGE gels (Invitrogen) for 3 h at 4 °C to increase separation between cleaved and intact SNAP-25.

Techniques: Staining, SDS Page, Translocation Assay, Molecular Weight