Extended Data Fig. 10: RPL12 regulates ribosome turnover in mice.
a, HEK293T cells were co-transfected with mRPL12-3×FLAG and HA-tagged mLC3s or mGABARAPs. After EBSS starvation for 1 hour, cell lysates were immunoprecipitated with anti-HA beads and analyzed by western blot with anti-FLAG antibodies. b, HEK293T cells were co-transfected with mRPL12-3×FLAG or mRPL12 P3N-E21L-3× FLAG, along with HA-tagged mLC3A, mLC3B, or mGABARAP. Following EBSS starvation for 1 h. Cell lysates were immunoprecipitated using anti-HA agarose beads and analyzed by western blot with an anti-FLAG antibody. c, GST pulldown assays were conducted using His-TF and His-TF-mRPL12 proteins, along with GST, GST-mLC3s, or GST-mGABARAPs from E. coli. d, GST pulldown assays were performed using His-TF, His-TF-mRPL12, or His-TF-mRPL12 P3N-E21L proteins with GST, GST-mLC3s, or GST-mGABARAP, all purified from E. coli. e, The expression levels of ribosomal and other indicated proteins in the mouse liver under starvation were assessed after intraperitoneal injection with mCherry-tagged rAAV-RPL12-WT or rAAV-RPL12 P3N-E21L (n = 3 mice). f, Degradation ratios from (e) were quantified. g, Total RNA from (e) was analyzed on formaldehyde agarose gels. h, rRNAs levels from (g) were quantified. i, A volcano plot showed MS signal changes in Rpl12 P3N-E21L mutant versus WT liver samples, with red dots (n = 27) indicating significantly changed components |log2FC | > log21.2 and P < 0.01. A two-sided Wald test was conducted without adjustment for multiple comparisons. j, R package DESeq2 (version 1.34.0) was used to identify differentially abundant component in MS from (i), selected by the threshold P < 0.01 and |log2FC | > log21.2. A two-sided Wald test was conducted without adjustment for multiple comparisons. k, l, Blood glucose (k) and hepatic glycogen concentrations (l) under starvation conditions were quantified. Actin and Tubulin served as the loading control. Blots and gels are representatives of three independent experiments. Statistical significance: ***P < 0.001, **P < 0.01, ns: no significance. P values were calculated by two-tailed Student’s t tests (f, h, k, l) and the data are presented as mean ± s.d. (n = 3 biological replicates). Exact P values, source numerical data and unprocessed blots/gels are provided in Source data.
