Hgh elisa kit

RBL cells were co-transfected with 2 μg of human growth hormone (hGH) secretion reporter plasmid [25 (link)] together with 18 μg of empty vector or various test plasmids (EGFPC2 vector alone or EGFP-tagged SNAP-23 cysteine mutants, SNAP-23 P119A mutant or SNAP-23 Cys⁻-MDR3_1–145 fusion construct). Transfected cells were triggered for exocytosis by cross-linking their high affinity FcεRI receptor using previously described method [7 (link)]. Briefly, subconfluent cells were sensitized overnight with DNP (dinitrophenol)-specific IgE (TIB-142 hybridoma culture supernatant) and IgE sensitized cells were either mock-stimulated (resting) with media alone or stimulated with 100 ng/ml DNP-BSA (Bovine serum albumin) for 45 min. The amount of hGH released into the culture supernatant and remaining in the cell lysates was determined using hGH ELISA kit (Roche Diagnostics) and expressed as a percentage of net hGH (stimulated-resting) released in the supernatant relative to total hGH in the cells.

The constructs coding for GFP-tagged mouse and human Sox4 and Stxbp6 were purchased from OriGene Technologies (Rockville MD, USA). The Y123C mutation was introduced into mouse Sox4 using QuikChange protocol (Agilent Technologies). INS-1 832/13 cells were transfected using Lipofectamine RNAiMAX (Life Technologies, Paisley, UK). The following siRNAs were used: ON-TARGETplus siRNA SMARTpool for Stxbp6 gene and ON-TARGETplus Non-Targeting Control siRNA (Thermo Scientific, Hemel Hempstead, UK). After 24h, the cells were cotransfected with human growth hormone (hGH) and either DsRed or GFP-tagged mouse Sox4 or Stxbp6 using GeneJuice (Merck Millipore, Nottingham, UK). Supernatants and cell pellets were collected and the amount of growth hormone was measured by using an hGH ELISA kit (Roche Diagnostics, West Sussex, UK). EndoC-βH2 cells (14 (link)) were transfected with Human specific ON-TARGETplus siRNA SMARTpools (Thermo Scientific, Hemel Hempstead, UK) for STXBP6 and SOX4 were used in siRNA knockdown experiments. siRNA and transfections were performed as described for INS1-832/13 cells. Quantitative analysis of gene expression was performed using QuantiFast SYBR Green PCR kit and gene specific QuantiTect Primer Assays (both from Qiagen). Expression was calculated using ΔCt method with GAPDH as a reference gene.