Journal: Nucleic Acids Research

Article Title: Loss of EZH2 results in precocious mammary gland development and activation of STAT5-dependent genes

doi: 10.1093/nar/gkv776

Figure Lengend Snippet: Expression of STAT5 target genes is enhanced in the absence of EZH2. ( A ) Heat map of 1181 genes expressed differentially in mammary tissue at day 13 of pregnancy (p13) in the presence and absence of EZH2 and in p18 wild type tissue. Genes expressed below 5 FPKM in E2KO tissue were removed. The heat map shows 398 genes activated more than 2-fold in E2KO tissue compared to wild type. ( B ) Pie charts showing overlap of up- or down-regulated genes in E2KO tissue with genes differentially expressed in wild type mice between p13 and p18. GO analysis of up- and down-regulated genes at p13 in E2KO tissue compared to wild type tissue. ( C ) Differentially expressed genes (DEGs) between p13 and p18 wild type tissue (left panel) and at p13 in the absence and presence of EZH2 (right panel). Three mice from each genotype were analyzed. FDR-adjusted P- value cutoff < 0.05. ( D ) Coverage plot (top) of normalized tags from RNA-seq shows differential expression throughout gene structure (bottom). Numbers indicate expression fold changes in the absence of EZH2 compared to control. ( E ) qRT-PCR results of Socs2, Wap and Csn2 mRNA levels at p13 are shown, confirming the RNA-seq data ( n = 6). Relative gene expression was obtained from β-actin.

Article Snippet: Quantitative PCR was performed using the TaqMan probe-based system (Socs2, Mm00850544_g1; Csn2, Mm00839664_m1; Wap, Mm00839913_m1 and mouse ACTB Endogenous Control on the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems)).

Techniques: Expressing, RNA Sequencing, Quantitative Proteomics, Control, Quantitative RT-PCR, Gene Expression

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae inhibited milk fat and protein synthesis in BMECs. (A–C) mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (D) DEGs heat maps. (E) Enrichment analysis of differentially expressed genes KEGG. (F) The protein levels of SREBP1 and β-casein were detected in BMECs. (G, H) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (I, J) mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (K) Typical images of BODIPY 493/503 staining in BMECs. * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative RT-PCR, Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae caused mitochondrial dysfunction in mammary gland and dyssynthesis of milk fat and protein. (A) Representative images of H&E staining. (B) The severity of mastitis was assessed by differences in histological score ( n = 6 cows per group) between the control and K. pneumoniae infection groups. (C–G) mRNA levels of TNF-α , IL-1β , IL-6, SREBP1 , and β-casein were detected by RT-qPCR method in mammary gland tissues (mean ± SEM, n = 6). (H) The protein levels of SREBP1, β-casein OPA1, MFN1, COX I, DRP1, and FIS1 were detected in mammary gland tissues. (I–O) Relative protein abundance of OPA1, MFN1, COX I, DRP1, FIS1, SREBP1, and β-casein were normalized to β-actin (mean ± SEM, n = 3). (P) Relative ATP levels (mean ± SEM, n = 6). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Staining, Control, Infection, Quantitative RT-PCR, Quantitative Proteomics

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: Mdivi-1 recovered K. pneumoniae -induced mitochondrial damage and dyssynthesis of milk fat and protein in BMECs. (A) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of OPA1, MFN1, COX I, DRP1, and FIS1. (B–F) Relative protein abundance of OPA1, MFN1, COX I, DRP1, and FIS1 were normalized to β-actin (mean ± SEM, n = 3). (G) Relative ATP levels (mean ± SEM, n = 3). (H) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of SREBP1 and β-casein. (I, J) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (K, L) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of SREBP1 and β-casein (mean ± SEM, n = 3). (M) Typical images of BODIPY 493/503 staining in BMECs. (N–P) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of TNF-α , IL-1β , and IL-6 (mean ± SEM, n = 3). (Q) Detection of the content of LDH (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: FNIP1 silencing alleviated K. pneumoniae -induced milk fat and protein dyssynthesis. (A) After FNIP1 silence, BMECs were infected with K. pneumoniae for 6 h to analyze the protein levels of SREBP1 and β-casein. (B, C) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (D, E) After FNIP1 silence, mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (F) Typical images of BODIPY 493/503 staining in BMECs. (G–I) After FNIP1 silence, mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Infection, Quantitative Proteomics, Quantitative RT-PCR, Staining

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

Journal: OMICS : a Journal of Integrative Biology

Article Title: Purification of the COP9 Signalosome Complex and Binding Partners from Human T Cells

doi: 10.1089/omi.2011.0158

Figure Lengend Snippet: Peptides of the CSN Subunits Identified by LC-MS

Article Snippet: The antibodies to CSN1 (A300-026A), CSN2 (A300-027A), CSN4 (A300-014A), CSN7b (A300-240A), and Cullin-4a (A300-738A) were obtained from Bethyl Laboratories (Montgomery, TX).

Techniques: Sequencing