Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Antibodies used (alphabetical order)

Article Snippet: ENaCβ , StressMarq Biosciences (SPC-404, Victoria, BC, Canada) , WB, 1:5,000.

Techniques: Generated

Journal: eLife

Article Title: Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity

doi: 10.7554/eLife.51404

Figure Lengend Snippet:

Article Snippet: Antibody , rabbit polyclonal anti-Na v 1.6 , Alomone , Cat # ASC-009 RRID: AB_2040202 , (1:200).

Techniques:

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Iron does not transcriptionally regulate ENaC alpha. The kidneys of 12-week-old WT or iron overloaded Hamp -/- mice were analyzed by real time PCR for gene expression of ENaC alpha. There was no significant difference in ENaC alpha gene expression of WT or spontaneously iron overloaded Hamp -/- mice under basal conditions ( A ). We confirmed that iron can regulate ENaC alpha gene transcription in mpkCCD cells, a cortical collecting duct cell line that is known to express ENaC alpha. 200 × 10 5 cells were serum-starved for 12 h and then cultured for 12 more hours in vehicle or FAC (100 uM). RNA was purified and then cDNA was synthesized. ENaC alpha gene expression was measured. Iron loading did not regulate ENaC alpha gene expression in mpkCCD cells ( B ), though it increased oxidative stress ( C ). One hundred uM FAC did not activate cell death program under the experimental conditions ( D ). Data from 2 independent experiments were pooled and analyzed. The statistical significance was determined by a 2-tailed Mann–Whitney test and is plotted as mean ± SEM. ** p < 0.001.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Synthesized, MANN-WHITNEY

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Immunohistochemistry analysis of ENaC alpha and MARCKS protein expression in Hamp -/- (KO) mice or wild-type (WT) mice. ( A ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in WT control mice. ( B ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in iron overloaded Hamp -/- mice. Data is representative of 3 mice in each group.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing

Journal:

Article Title:

doi:

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:

Article Title:

doi:

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

Journal:

Article Title:

doi:

Figure Lengend Snippet: SNPs Upstream of the CHRNB4 Gene Are Associated with CHRNB4 Gene Expression in Islets from Human Donors

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, Significance Assay

Journal: International journal of molecular sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells.

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Figure 3. Membrane colocalization and overlapping between α3β4 nAChR (red) and DBH (green) after secretion stimulus: in each image we individually selected several regions of interest (ROIs) (a,b) and proceeded to analyze them using ImageJ JACoP complement (See Section 4.6), obtaining the values of the Pearson’s and Manders coefficients, whose averages are shown in Table 1, as well as their corresponding scatterplot graphs, whose examples are shown (d). The averaged Pearson’s coeficients for 50% threshold images show a significant colocalization between endogenous α3β4 nAChRs structures and the DBH sites in both stimulation conditions to those obtained for randomly simulated α3β4 nAChRs structures (Table 1 (c)). Furthermore, the averaged coefficients obtained for the stimulus with ACh are significantly higher than those obtained when stimulating with a high K+ (p value *** < 0.0001; Table 1). (d) A scatterplot or fluorogram for pixel colocalization of the red channel (AChR structures) and green channel (DBH) comparing three examples of the images obtained in each of the three analyzed conditions. Bars represent 1 µm.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Membrane