anti cops2  (Bethyl)

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: Genotypes of offspring from Csn2 WT/K70E intercrosses.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques:

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a Body weight measurement of wild-type and Csn2 WT/K70E (Het) mice at the indicated age (*** p < 0.001, calculated by two-way repeated measures ANOVA testing genotype × time effect). Data are presented as mean ± SEM. b Feeding and fasting (20 h) glucose levels ( n = 5). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. c , d Feeding and fasting (16 h) insulin ( c ) and C-peptide ( d ) levels ( n = 5). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. e Intraperitoneal glucose tolerance test [ n = 5 (WT), or 6 (Het), * p < 0.05, ** p < 0.01, calculated by two-tailed Student’s t test.]. Data are presented as mean ± SD. f Intraperitoneal insulin tolerance test (ITT; n = 5 (WT), or 6 (Het), * p = 0.012, calculated by two-way repeated measures ANOVA testing genotype × time effect). Data are presented as mean ± SD. 100% blood glucose means 6.37 ± 0.6 mM for WT and 7.27 ± 0.8 mM for Het. g , h Effect of streptozocin (STZ) treatment (50 mg/kg, 4 days) on serum insulin levels ( g ) and insulin tolerance test ( h ) of wild-type and Csn2 WT/K70E (Het) mice, ( n = 5). Data are presented as mean ± SD. 100% blood glucose means 14.0 ± 5.4 mM for WT and 11.2 ± 2.2 mM for Het. P values were calculated by two-tailed Student’s t test. i Glucose-stimulated insulin secretion in mice [ n = 4 (WT), or 5 (Het), ** p < 0.01, calculated by two-tailed Student’s t test.]. Data are presented as mean ± SEM. j Glucose-stimulated insulin secretion in isolated pancreatic islets ( n = 3, *** p < 0.001, calculated by two-tailed Student’s t test.). Data are presented as mean ± SEM. k Basal and secretagogue-stimulated insulin secretion (1 h) in isolated islets ( n = 3). Data are presented as mean ± SD. Islets from Csn2 WT/K70E mice secrete more insulin with l -arginine stimulation, suggesting that altered membrane potential or Ca 2+ flux are not major causes of their enhanced insulin secretion. P values were calculated by two-tailed Student’s t test.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Two Tailed Test, Isolation, Membrane

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a , b H&E staining-based morphometric analysis of pancreatic islets from wild-type and Csn2 WT/K70E mice. Number of islets per pancreas ( a ) and average islet area ( b ) was measured by examining 20 slides with every five slides in between, covering the whole pancreas ( n = 5, n.s. not significant). Data are presented as mean ± SEM. Scare bars, 1.5 μm ( a ) and 0.4 μm ( b ). c Immunofluorescence staining of insulin and glucagon in pancreatic islets from wild-type and Csn2 WT/K70E mice. Scare bars, 0.2 μm. Lower panel: percentage of α or β cells in an islet is measured based on the area of green (anti-glucagon, α cells) and red fluorescence (anti-insulin, β cells; n = 8, n.s. not significant). Data are presented as mean ± SEM. Right panel: quantification of the immunofluorescence intensity of insulin staining ( n = 8). P values were calculated by two-tailed Student’s t test. d , e Average islet cytoplasmic Ca 2+ responses to depolarization induced by 20 mM glucose ( d ) or 25 mM KCl ( e ), assayed with Fura2AM ( n = 5, n.s. not significant). Data are presented as mean ± SEM. f Total insulin content of pancreatic islet measured using ELISA assay ( n = 10). P values were calculated by two-tailed Student’s t test. g Levels of Ins1 , Ins2, Sytl3 , and Exoc6 transcripts from pancreas measured by real-time PCR ( n = 10). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test. h Representative electron micrographs of pancreatic islets showing β cell insulin granules. Dotted lines: plasma membrane (PM). Docked granules are marked by arrow heads. Scale bars, 0.5 μm ( n = 6). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test. i GSIS of wild-type and Het islets measured as percentage of insulin secretion. Islets were lysed after GSIS to determine total insulin content ( n = 5). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Staining, Immunofluorescence, Fluorescence, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a Effect of MLN4924 treatment (2 μM, 2 h) on basal and glucose-stimulated insulin secretion by wild-type and Csn2 WT/K70E islets ( n = 6, *** p < 0.001, calculated by two-way ANOVA testing the effect of 2.8 or 16.8 mM glucose in the presence of MLN4924, the other p values were calculated by two-tailed Student’s t test). Data are presented as mean ± SEM. b , c Mice serum insulin levels with/without MLN4924 treatment (15 mg/kg, twice weekly for 6 weeks). Insulin levels were measured at the same time of the day to minimize fluctuation. ( n = 6, ** p = 0.001, calculated by two-way repeated measures ANOVA testing genotype × time effect for Het mice). Data are presented as mean ± SEM. c Mice body weight with/without MLN4924 treatment (15 mg/kg, twice weekly for 6 weeks; n = 6, *** p < 0.001, calculated by two-way repeated measures ANOVA testing genotype × time effect for Het mice). Data are presented as mean ± SEM. d Levels of total and neddylated Cullins, COP1, and CRL4 substrates in the pancreas after 6 h fasting to normalize variation in blood glucose levels. e Ex vivo, lentiviral shRNA-based knockdown screening for CRL4 adaptors whose depletion alters islet insulin secretion. Quantified values of insulin secretion were normalize by that of Ctrl islets and presented in Log 2 scale. f Effect of adenovirus-mediated in-islet COP1 knockdown on basal and glucose-stimulated insulin secretion ( n = 3, *** p < 0.001, calculated by two-way ANOVA testing the effect of 2.8/16.8 mM glucose in the presence of shCOP1, the other p values were calculated by two-tailed Student’s t test). Data are presented as mean ± SEM. Inset: western blot showing efficient COP1 knockdown. Arrow indicates COP1 band. An asterisk (*) indicates nonspecific band. g Levels of ETV4 and ETV5 in MIN6 cells with COP1 (left) or Det1 (right) knockdown using two independent shRNA. h Levels of ETV5 in MIN6 cells with Myc-Det1 and HA-COP1 single or double overexpression. i Levels of ETV5 in wild-type and Csn2 WT/K70E islets with/without MLN4924 treatment (2 μM, 2 h). j Levels of ubiquitylated ETV5 enriched by GST-TUBE pulldown. k Effect of CSN2 knockdown on ETV5 ubiquitylation in MIN6 cells. l Effect of adenovirus-mediated in-islet ETV5 knockdown on insulin secretion ( n = 3). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. m Effect of ETV5 overexpression on insulin secretion ( n = 4). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. n Effect of adenovirus-mediated in-islet ETV5 knockdown on mRNA levels of ETV5 transcriptional targets ( n = 3). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. o Effect of ETV5 overexpression on mRNA levels of ETV5 transcriptional targets ( n = 3). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. p Effect of Sytl3 and Exoc6 overexpression on insulin secretion ( n = 3). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Two Tailed Test, Ex Vivo, shRNA, Knockdown, Western Blot, Over Expression

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a Structural models illustrating steric incompatibility between COP1 and CSN. For modeling details, see “Methods”. b Effect of COP1 and DET1 overexpression, individually or combined, on CRL–CSN complex formation in MIN6 cells. c , d CRL4–COP1 complex formation detected by COP1 co-immunoprecipitation with Cul4A ( c ) and Cul4B ( d ) from WT and Csn2 WT/K70E pancreas lysates. e Effect of adding IP 6 (20 μM) into MIN6 cell lysates on CRL4B COP1 complex formation. f Glucose fasting (2 h) increases CSN3-Cul4B co-ip in MIN6 cells, which is reversed by replacement with glucose-proficient medium (1 h). g Glucose fasting (2 h) decreases Cul4B-CSN3 co-ip in MIN6 cells, which is reversed by replacement with glucose-proficient medium (1 h).

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: CRL4-sequestration by CSN is compromised by CSN2-K70E mutation, or hyperglycemia elicited by nutrient oversupply. This leads to the assembly of CRL4 COP1 E3 ligase to promote ETV5 degradation, which acts as a transcriptional “brake” suppressing congenital hyperinsulinemia and consequent obesity/diabetes.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Mutagenesis