Journal: Nucleic Acids Research

Article Title: The RNA-binding protein PRRC2B preserves 5′ TOP mRNA during starvation to maintain ribosome biogenesis during nutrient recovery

doi: 10.1093/nar/gkaf1334

Figure Lengend Snippet: PRRC2B L and PRRC2B S have different protein interactomes. ( A-D ) HEK 293T cells were transiently transfected with pcDNA plasmids expressing HA-mCherry as a control, HA-tagged PRRC2B L or PRRC2B S . Twenty-four hours after transfection, HA–protein complexes were co-immunoprecipitated and analyzed by Mass Spec. ( A, B ) Volcano plot of the fold-ratio of the abundance of the detected proteins in PRRC2B L ( A ) or PRRC2B S ( B ) versus control IP samples, versus their significance expressed as -log 10 P -value. Top proteins with significantly increased abundance are indicated in red for each isoform, highlighting translation initiation factors. Pie charts beneath graphs indicate the distribution of interacting proteins within the indicated categories. ( C ) Venn diagram depicting overlap between PRRC2B L and PRRC2B S interactomes. ( D ) String network analysis of a set of 69 proteins that show significant enrichment (FC > 2, p < 0.05) in PRRC2B L interactome compared to PRRC2B S interactome. ( E, F ) HEK 293T cells transiently transfected with pcDNA plasmids expressing HA-mCherry as a control, HA-tagged PRRC2B L or PRRC2B S were immunoprecipitated with anti-HA antibodies in the absence or presence of 100mg/ml RNase A, and lysates and IPs were subjected to western blot analysis for the indicated proteins. Tubulin was used as a loading control. Red asterisks in ( F ) denote a degradation product of PRRC2B L , which increases during the IP procedure. Shown are representative blots of 4 ( E ) or 2 ( F ) independent experiments.

Article Snippet: Plasmids for expression of exogenous proteins were introduced to cells by the standard calcium-phosphate transfection method or Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol.

Techniques: Transfection, Expressing, Control, Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: bioRxiv

Article Title: Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

doi: 10.1101/2025.11.20.689563

Figure Lengend Snippet: (A) MMEJ reporter assay in K562 cells expressing the indicated sgRNAs. Data are presented as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001). (B) Immunoblot of endogenous POLQ, SMARCB1, and GAPDH in WT RPE cells with empty vector, SMARCB1 -KO RPE cells with empty vector, and SMARCB1 -KO RPE cells reconstituted with WT SMARCB1. (C) MMEJ reporter assay in HEK293T WT and SMARCB1 -KO cells with or without 3xFLAG POLQ transfection. (D) Immunoblot of Flag-tagged POLQ expression in HEK293T WT and SMARCB1 -KO cells with or without 3xFLAG POLQ transfection. (E) Immunoblot of endogenous POLQ, SMARCB1, and GAPDH in WT RPE cells, SMARCB1 -KO RPE cells, and rhabdoid tumor cell lines (G401, A204). (F) Representative immunofluorescence images showing nuclear foci of HA–tagged endogenous POLQ in U2OS cells expressing sg NT or sg SMARCB1 . Quantification of HA– POLQ foci per cell is shown below. Data are shown as individual data points representing the number of HA-POLQ foci per nucleus; the mean is indicated by a horizontal bar. Statistical significance was calculated using Mann-Whitney U test (****, P < 0.0001). (G) Percentage somatic SNVs attributed to COSMIC single base substitution signatures (v3.3) for Rhabdoid Tumors (n = 56), Wilms Tumor (n = 81), and Neuroblastoma (n = 135). Data are shown as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (****, P < 0.0001).

Article Snippet: Lentiviral particles were generated by transfecting HEK293T cells with a mixture of the lentiviral transfer plasmid, the packaging plasmid psPAX2, and the envelope plasmid pMD2.G using a calcium phosphate transfection kit (Takara Bio, #631312).

Techniques: Reporter Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, Immunofluorescence, MANN-WHITNEY, Wilms Tumor Assay