Fig. 5: SL–protein interactomics reveals a role for Tmed2 in proinsulin processing.
From: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis
a, Experimental setup for identification of SBPs in a SILAC-based approach. pacSph treatment of Sgpl1ΔIns1E and CerS2:Sgpl1ΔIns1E cells differentially labelled with stable isotopes allows crosslinking of SL-protein complexes by UV irradiation (with omission of UV irradiation as a control condition), followed by cell lysis and conjugation of biotin to the SL-protein complexes. After Streptavidin-based pull-down, SBPs can be identified and quantified in the same MS run by the differing peptide mass due to SILAC isotope labelling. b, Volcano plot showing log2 fold change of proteins pulled down from pacSph-treated Sgpl1ΔIns1E (+UV) versus Sgpl1ΔIns1E (−UV) cells plotted against the −log10 P values of a one-sample two-sided t-test against 0. Proteins with log2 fold change >1 and a BH-FDR <0.05 are regarded as SBPs (n = 4 independent experiments). c, Volcano plot showing log2 fold change of SBPs identified in b (Supplementary Fig. 3e) and pulled down from pacSph-treated CerS2:Sgpl1ΔIns1E (+UV) versus Sgpl1ΔIns1E (+UV) cells plotted against the −log10 P values of a two-sample two-sided equal variance t-test (n = 4 independent experiments). SBPs with a fold change >1.5 and a BH-FDR <0.05 were regarded as Cers2-dependent SBPs. Fold enrichment was 6.55 and FDR-corrected P value was 3.12−10 for GO term ‘endoplasmic reticulum’. d, pacSph pull-down of endogenous Tmed2 in SgplΔIns1E and Sgpl1:CerS2ΔIns1E cells (n = 4 independent experiments); exemplary immunoblot (right) and quantification (left). Eluate intensities were normalized to respective input intensities. e, Relative mRNA expression of Tmed1, Tmed2 and Pcsk1 in murine pseudoislets transfected with control siRNA or siRNA against Tmed1, Tmed2 or both. n = 4 independent experiments. f–i, Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in pseudoislets transfected with siRNA as described in Extended Data Fig. 8e; n = 3 independent experiments. f, Representative immunoblot. g, Quantification of Pcsk1. h, Quantification of Pro-Pcsk1. i, Ratio of Pcsk1 to Pro-Pcsk1. j–l, Insulin content (j), proinsulin content (k) and ratio of insulin to proinsulin (l) in pseudoislets transfected with siRNA as described in Extended Data Fig. 8e determined via ELISA (n = 8 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test (d) and repeated measures one-way ANOVA with Tukey’s multiple comparisons test (e and g–l). In e, ANOVA was performed for each mRNA target individually. P values are stated in each figure. Data points in d, e and g–l represent individual experiments. Bar graphs represent mean ± s.e.m. For one experiment in j–l, the mean of five replicates, consisting of nine pseudoislets, respectively, was plotted per condition. Stain-free signal was used for normalization of immunoblots in g–i. Source numerical data and unprocessed blots are available in source data.
