Fig 1: L1 bodies (LBs) are large cytoplasmic compartments enriched in ORF1p, L1 mRNA, and ribosomes.(A–E) Electron micrographs exemplifying the formation of LBs in Mael-/- spermatocytes. Boxed areas in A and B identify small cytoplasmic aggregates magnified in A’ and B’, respectively. Boxed area in C identifies an intermediate size LB magnified in C’. D and E show more prominent LBs, partially surrounded by a bilayer membrane (red arrows). Nuc: nucleus. Cyto: cytoplasm. NE: nuclear envelope. (F) Immunofluorescence staining of L1 ORF1p on Mael-/- spermatocytes shows ORF1p accumulation in LBs (left panel, magenta). 3D reconstruction of LBs (magenta) and nuclei (blue) (middle and right panels); numbers [1–5] are used to label corresponding cells. Scale bars: 5 µm. (G) HCR RNA-FISH of L1 RNA (green) and immunofluorescence staining of L1 ORF1p (magenta) on Mael+/- and Mael-/- testis sections. Scale bars: 5 µm. (H) Double immunofluorescence staining of ribosomal protein S6 (RPS6, top two rows, green) or L28 (RPL28, bottom two rows, green) and L1 ORF1p (magenta) showing their colocalization in LBs. Dotted white lines mark seminiferous tubule boundaries. Scale bars: 15 µm. See also S1 Fig.
Fig 2: Cytoplasmic NFATc3 facilitates VSMC contractile-to-synthetic phenotype switching by promoting global protein synthesis. (A) Representative images and quantification of NFATc3 and ACTA2 expression using immunofluorescence staining in MASMCs isolated from mice treated with vehicle and BAPN for 28 days (n = 6). (B) NFATc3-associated protein subcellular localization based on LC–MS/MS. (C) Gene Ontology analysis of NFATc3-associated proteins identified using LC–MS/MS. (D) Polysome profiling shows NFATc3 distribution in MASMCs using sucrose density gradient centrifugation (n = 3). Western blot of NFATc3 in the elution profiles. (E) Representative Western blot images and quantification of puromycin incorporation assays from MASMCs isolated from Nfatc3-KIfl/fl and Nfatc3smcKI mice (n = 4). (F) HPG incorporation assay in VSMCs isolated from the aortas of Nfatc3-KIfl/fl and Nfatc3smcKI mice treated with BAPN for 28 days (n = 4). (G) Western blot and quantification of ACTA2, CNN1, SM22α, and OPN in MASMCs treated with PDGF-BB for 24 h before a 12-h CHX treatment (n = 4). (H) Representative Western blot images and quantification of ACTA2, CNN1, SM22α, and OPN in MASMCs isolated from aortas of Nfatc3-KIfl/fl and Nfatc3smcKI mice and treated with PDGF-BB (20 μg/L) for 24 h before a 12-h CHX (50 μmol/L) treatment (n = 4). (I) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. Western blot of RPL28 and RPS6 levels. Data are presented as mean ± SD. (E) Unpaired Student’s t-test; two-tailed P-values. (G) Two-way ANOVA with Tukey’s correction; adjusted P-values. (H) One-way ANOVA with Tukey’s correction; adjusted P-values.
Supplier Page from Abcam for Anti-RPL28 antibody