pcsk1-myc-ddk  (OriGene)

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Verification of loss of CerS2 in CerS2 ΔIns1E cells by immunoblot. Left: representative immunoblot. Right: quantification of Cers2 signals ( n = 8 independent experiments). b , Representative immunostaining (left) and quantification (right) of ER marker PDI in control and CerS2 ΔIns1E cells ( n = 36 control versus 28 CerS2 ΔIns1E well sites from one experiment). Scale bar, 10 μm. c , Quantification of insulin content in control and CerS2 ΔIns1E cells at low (2 mM) and high (25 mM) glucose levels ( n = 5 independent experiments). d , Experimental design and results for proteome analyses in control and CerS2 ΔIns1E cells. e , Volcano plot showing log 2 fold change of proteins between CerS2 ΔIns1E and control cells plotted against the −log 10 P value of a two-sided paired Student’s t -test. BH-FDR <0.05 and fold change >1.5 was used as significance cut-offs ( n = 3 control versus 3 CerS2 ΔIns1E samples collected in three independent experiments). f , g , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in islets of control and CerS2 ΔBKO mice. f , Representative immunoblot. Each lane represents islets of one individual mouse. g , Quantification of Pro-Pcsk1 (left) and Pcsk1 (middle) protein levels and ratio of Pcsk1/Pro-Pcsk1 (right; n = islets of six control versus six CerS2 ΔBKO mice). h , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in islets of 12-week-old control and db/db.BKS mice. Quantification of Pro-Pcsk1 (left) and Pcsk1 (middle) protein levels and ratio of Pcsk1/Pro-Pcsk1 (right; n = islets of six control versus six db/db.BKS mice). Representative immunoblot is shown in Extended Data Fig. . i , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels in islets of 12-week-old control and ob/ob.B6 mice. Quantification of Pro-Pcsk1 (left) and Pcsk1 (middle) protein levels and ratio of Pcsk1/Pro-Pcsk1 (right; n = islets of six control versus six ob/ob.B6 mice). Representative immunoblot is shown in Extended Data Fig. . Statistical analysis was performed using a two-sided Student’s t -test ( a , b , g , h and i ) and two-way ANOVA with Sidak’s multiple comparisons test ( c ). P values are stated in each figure. Bar graphs represent mean ( c ) or mean ± s.e.m. ( a , b , g , h and i ). Connecting lines indicate both samples are from one experiment. Data points in b represent individual well sites. Data points in a and c represent independent experiments. Data points in g – i represent islets from individual mice. Stain-free signal was used for normalization of all immunoblots. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Western Blot, Immunostaining, Marker, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Quantification of Pcsk1 levels in control and CerS2 ΔIns1E cells by CrispR-verified antibody (Supplementary Fig. , Cell Signaling #11914, discontinued). Left, representative immunoblot. Right, quantification of Pcsk1 signals ( n = 5 independent experiments). b , Quantification of Pcsk1 protein levels in control and CerS2 BKO islets by CrispR-verified antibody (Supplementary Fig. , Cell Signaling #11914, discontinued). Left, representative immunoblot. Right, quantification of Pcsk1 signals ( n = 8 independent experiments). c , Quantification of Pcsk1 mRNA levels in islets from male control and CerS2 BKO mice by qPCR ( n = 5 independent experiments). d , Representative immunoblots to Fig. . e , Representative immunoblots to Fig. . f, g , Quantification of mRNA levels of Pcsk1 and various CerS in islets of 12 week old control and db/db.BKS mice (f, n = 4 control and 4 db.db/BKS islet samples) and islets of 12 week old control and ob/ob.B6 mice (g, n = 4 control and 6 ob/ob.B6 islets samples). Statistical analysis was performed using a paired two-sided Student’s t -test (a, b), two-sided Student’s t -test (c) and two-sided multiple t -tests with Holm-Sidak correction (f, g). P -values are stated in each figure. Bar graphs represent means (a, b) or means + s.e.m. (c, f, g). Connecting lines indicate both samples are from one experiment. Data points represent independent experiments (a), islets from individual mice (c) or individual islet samples (f, g). Stain-Free signal was used for normalization of all immunoblots. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: CRISPR, Western Blot, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Immunoblot detection of human CerS6 (hCerS6) and Tmed2 protein levels in INS1E cells after overexpression of hCerS6 via adenovirus for 48 h (representative immunoblot). b , Ins1E cell counts after 48 hours of infection with a control adenovirus or hCerS6-expressing adenovirus ( n = 3 independent experiments). A reduction of cell counts is in line with the ability of CerS6 overexpression to induce apoptosis in several cell types. c , Immunoblot detection of Pro-Pcsk1 and Pcsk1 protein levels (Cell Signaling #18030) in Ins1E cells infected with a control adenovirus or hCerS6-expressing adenovirus for 48 h (representative immunoblot). d-g , Quantification of Tmed2 (d), Pcsk1 (e), Pro-Pcsk1 signals (f) and Pcsk1/ Pro-Pcsk1 ratio (g) from 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (b, d-g). Data points in (b, d-g) represent independent experiments. Bar graphs in (b, d-g) represent means + s.e.m. Stain-Free signal was used for normalization of all immunoblots. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Western Blot, Over Expression, Infection, Expressing, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Experimental setup for identification of SBPs in a SILAC-based approach. pacSph treatment of Sgpl1 ΔIns1E and CerS2:Sgpl1 ΔIns1E 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 log 2 fold change of proteins pulled down from pacSph-treated Sgpl1 ΔIns1E (+UV) versus Sgpl1 ΔIns1E (−UV) cells plotted against the −log 10 P values of a one-sample two-sided t -test against 0. Proteins with log 2 fold change >1 and a BH-FDR <0.05 are regarded as SBPs ( n = 4 independent experiments). c , Volcano plot showing log 2 fold change of SBPs identified in b (Supplementary Fig. ) and pulled down from pacSph-treated CerS2:Sgpl1 ΔIns1E (+UV) versus Sgpl1 ΔIns1E (+UV) cells plotted against the −log 10 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 Sgpl ΔIns1E and Sgpl1:CerS2 ΔIns1E 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. ; 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. 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.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Irradiation, Lysis, Conjugation Assay, Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Top 20 SBPs identified in Fig. according to p -values and a log 2 fold change > 3. b , Verification of Bst2 and Fxyd6 as SBPs by overexpression of DDK-tagged variants in Sgpl1 ΔIns1E cells followed by pacSph-pulldown; representative immunoblots (left) and quantification (right). Eluate band intensities were normalized to input bands and +UV samples were set to 1 ( n = 4 independent experiments). c , Verification of Tmed1 as SBP as described in (b); representative immunoblot (left) and quantification (right). n = 3 independent experiments. d , Immunoblot detection of Tmed2 protein levels in Sgpl1 ΔIns1E and Cers2:Sgpl1 ΔIns1E cells. Representative immunoblot showing 3 replicates per genotype (left) and quantification (right). n = 3 independent experiments with 3 replicates per genotype, respectively. e , Immunoblot detection of Tmed2 protein levels in islets of 6 control and 6 Cers2 ΔBKO mice. Representative immunoblot (left) and quantification (right). f , Immunoblot detection of Tmed2 protein levels in islets of 6 control and 6 ob/ob.B6 mice at week 12. Representative immunoblot (left) and quantification (right). g , Immunoblot detection of Tmed2 protein levels in islets of 6 control and 6 db/db.BKS mice at week 12. Representative immunoblot (left) and quantification (right). Statistical analysis was performed using a one sample t -test against 1 (b, c) or Student’s t -test (d-g). P -values are stated in each figure. Bar graphs represent means + s.e.m. Data points in (b, c and d) represent individual experiments. Data points in (e-g) represent islets from individual mice. Stain-Free signal was used for normalization of all immunoblots, except for (b) and (c). Stain-Free images of (e), (f) and (g) were reproduced from Fig. , Extended Data Fig. , as the same PVDF membranes were used for detection of Pro-Pcsk1, Pcsk1 and Tmed2, respectively. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Over Expression, Western Blot, Staining

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Double cut CrispR/Cas9 knockout strategy for Tmed2 in Ins1E cells. b, c , Relative mRNA expression of various Tmed family members (b) and beta cell identity markers (c) in wildtype Ins1E, control and Tmed2 ΔIns1E cells. Wildtype Ins1E samples were set to 1 (dotted line). n = 3 independent experiments. Note that potentially as a sign of attempted compensation, Pcsk1 mRNA levels are increased in Tmed2-deficient Ins1E cells. d , Representative immunoblot analysis of Tmed2 protein expression in 3 control vs. 3 Tmed2 ΔIns1E replicate lysates. e , Insulin content in control and Tmed2 ΔIns1E cells determined via ELISA. n = 3 independent experiments with 3 replicates per genotype, respectively. Statistical analysis was performed using multiple two-sided t -tests with Holm-Sidak correction (b and c) and a two-sided Student’s t -test (e). Data points represent independent experiments. Bar graphs represent means + s.e.m. The control and Tmed2 ΔIns1E cells are pools of individual monoclonal cell lines, respectively (8 monoclonal control cell lines and 3 monoclonal Tmed2 ΔIns1E cell lines were used for pooling). Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: CRISPR, Knock-Out, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Cell Biology

Article Title: Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

doi: 10.1038/s41556-022-01027-2

Figure Lengend Snippet: a , Co-immunoprecipitation (Co-IP) of co-overexpressed Tmed2-V5 and Pro-Pcsk1/Pcsk1-DDK in Ins1E cells. Representative immunoblot (left) and quantification of three replicate experiments (right). As Ctrl-plasmid, the promotorless pNL1.3 from Promega (N1021) was used. b , Representative confocal images for co-localization of overexpressed Tmed2-V5 and Pro-/Pcsk1-DDK in Ins1E cells. Green, SytoxGreen as nucleus marker; red, Pro-/Pcsk1-DDK; blue, Tmed2-V5. Scale bar, 5 µm. c, d , Quantification of overlap of Pro-/Pcsk1-DDK with Tmed2-V5 (c) and Tmed2-V5 with Pro-/Pcsk1-DDK (d) in control and CerS2 ΔIns1E cells. n = 2 independent experiments; only one experiment shown. e-j , Overlap of ER-marker PDI and Golgi-Marker TGN46 with Tmed2-V5, Pro-/Pcsk1-DDK (allowing detection of both Pro-Pcsk1 as well as mature Pcsk1) and Pro-Pcsk1 (only allowing detection of the immature Pro-Pcsk1 protein) after overexpression in control and CerS2 ΔIns1E cells. n = 3 independent experiments. Statistical analysis was performed using a paired two-sided Student’s t -test (a) and unpaired two-sided Students t -tests (c-j). Data points represent replicate experiments (a) and individually quantified cells (c-d) or well sites (e-j). Bar graphs represent means + s.e.m. Source numerical data and unprocessed blots are available in source data.

Article Snippet: To determine overlap of Tmed2 and Pcsk1 with ER and Golgi, we used the following plasmids: Tmed2-Myc-DDK (Origene, #RC206849), Pcsk1-Myc-DDK (DDK-tag will be cleaved off during maturation into enzymatically active Pcsk1; Origene, #MR225451) and the Pro-/Pcsk1-DDK plasmid described above.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Marker, Over Expression