anti-nicotinic acetylcholine receptor beta2 (chrnb2) (extracellular) antibody  (Alomone Labs)

Journal: Cell reports

Article Title: MafA-Controlled Nicotinic Receptor Expression Is Essential for Insulin Secretion and Is Impaired in Patients with Type 2 Diabetes

doi: 10.1016/j.celrep.2016.02.002

Figure Lengend Snippet: (A–H) Immunohistochemistry of MafA WT and Maf A RIP islets to show expression of CHRNA4 (A and B; green), CHRNA5 (C and D; green), CHRNB2 (E and F; green), and CHRNB4 (G and H; green). β cells are stained for insulin (red) and nuclei (DAPI; blue); scale bar represents 20 µm. (I) qPCR amplification of Chrn upstream sequences after immunoprecipitation of βTC-6 chromatin with a MAFA or rabbit IgG antibody are presented as % input. Differences in percent input for IgG reflect variations in primer efficiencies. n = 4. (J) Induction of luciferase reporter activity of ChrnB2 (pB2LUC) and ChrnB4 (pB4LUC) luciferase reporter constructs upon co-transfection with MAFA. Empty vector control (pGl2b and pFOX) is set to one; n = 3 or 4. (K) qPCR measurements of Chrn expression levels in MafA siRNA-treated βTC6 cells; n ≥ 3. Data were normalized to the geomean of HPRT and β- actin mRNA levels. (L) Dynamic insulin secretion of MafA WT and MafA RIP islets stimulated with 10 mM glucose (G) and 100 µM nicotine (NIC), 100 µM nicotine + 100 µM oxotremorine (NIC+OXO), and 100 µM oxotremorine (OXO). The transient decrease in insulin secretion upon NIC treatment in MafA WT islets is marked by a solid arrow. The biphasic insulin secretion induced by NIC+OXO treatment is indicated by a dashed arrow and a dotted arrow; n = 8. (M) Dynamic insulin secretion of wild-type islets with 1 or 10 µM acetylcholine (n ≥ 5). Acetylcholine treatment is illustrated by black lines. Data are mean ± SEM and were analyzed using one-way ANOVA and Tukey multiple comparison tests (J) or paired t test (I and K). *p < 0.05 and **p < 0.01. See for validation of the nicotinic receptor antibodies.

Article Snippet: Primary antibodies were α-CHRNB2 antibody (1:200; no. ANC-012; Alomone Labs), α-ADRA2A antibody (1:500; no. A271; Sigma-Aldrich), α-CHRNA5 antibody (1:400; no. NBP1–69122; Novus Biologicals), α-CHRNA4 antibody (1:500; no. ANC-004; Alomone Labs), α-CHRNB4 antibody (1:500; no. ANC-014; Alomone Labs), and monoclonal mouse α-pactin antibody (1:2,000; Sigma).

Techniques: Immunohistochemistry, Expressing, Staining, Amplification, Immunoprecipitation, Luciferase, Activity Assay, Construct, Cotransfection, Plasmid Preparation

Journal: Cell reports

Article Title: MafA-Controlled Nicotinic Receptor Expression Is Essential for Insulin Secretion and Is Impaired in Patients with Type 2 Diabetes

doi: 10.1016/j.celrep.2016.02.002

Figure Lengend Snippet: (A) Regional plot of the CHRNB4 gene showing the presence of a cluster of SNPs that infer low expression of CHRNB4 in human islets. The leading SNP (rs12910237) is indicated in purple. (B) Effect of alternate rs12910237 allele copy numbers on CHRNB4 transcript levels in islets from human donors; n = 89 (p = 1.98E–05). (C) Overview of the CHRNB4 upstream region containing SNPs affecting islet gene transcription and the risk for developing type 2 diabetes. MAF and other transcription-factor-binding sites and active islet enhancer regions are shown . Transcriptional activity in the β cell line β-TC6 and activation by MafA is indicated. (D–G) Analysis of RNA-seq data from human donor islets to show the correlation between CHRNB2 expression and MAFA and MAFB transcript levels, insulin secretion (stimulatory index), and HbA1c levels. (H–K) Analysis of RNA-seq data from human donor islets to show the correlation between CHRNB4, MAFA , and MAFB ; stimulatory index; and HbA1c levels. Experiments were analyzed with linear regression and Pearson correlation analysis; p values are indicated in the respective graphs; n = 131. (L) siRNA-mediated knockdown of MAFA in EndoC-βH1 cells, showing the effect on mRNA levels of CHRNB2 (p = 0.04), CHRNB4 (p = 0.05), and ADRA2A (p = 0.055); n = 3 or 4. (M) Effect of pCMVMafA (MafA) expression on luciferase (Luc) activities of human CHRNB4 upstream reporter constructs (p-3.7kbLUC and p-7.5kbLUC) spanning sequences that contain identified risk(R) and corresponding non-risk alleles (NR) for rs12910237 or rs922691 and islet enhancer regions. Activity was assessed in HEK293 cells, which do not have endogenous MAFA activity. n = 4. Data are mean ± SEM and were analyzed using one-way ANOVA and Tukey multiple comparison tests (M) and Student’s t test (L). *p < 0.05; **p < 0.01; ****p < 0.001. Abbreviations: T2D, type 2 diabetes; TF, transcription factor. See also for additional correlation and transcriptional data.

Article Snippet: Primary antibodies were α-CHRNB2 antibody (1:200; no. ANC-012; Alomone Labs), α-ADRA2A antibody (1:500; no. A271; Sigma-Aldrich), α-CHRNA5 antibody (1:400; no. NBP1–69122; Novus Biologicals), α-CHRNA4 antibody (1:500; no. ANC-004; Alomone Labs), α-CHRNB4 antibody (1:500; no. ANC-014; Alomone Labs), and monoclonal mouse α-pactin antibody (1:2,000; Sigma).

Techniques: Expressing, Binding Assay, Activity Assay, Activation Assay, RNA Sequencing Assay, Luciferase, Construct