l glutamine  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: ?-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine *

doi: 10.1074/jbc.M110.127613

Figure Lengend Snippet: siRNA knockdown of α-AP-2 reduces CFTR endocytosis. A , HEK 293 cells expressing CFTR were incubated overnight in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and allowed to grow to 70% confluency. The cells were transfected with α-AP-2 or scrambled siRNA and analyzed by Western blot at 72 h to detect α-AP-2 and β-actin ( A ). B , cell surface proteins were labeled using NHS SS-biotin and allowed to internalize for 1 and 5 min at 37 °C. Following MESNA treatment, the cells were lysed, surface biotinylated proteins were bound to streptavidin-agarose beads, and CFTR was detected by Western blot. C , the percentage of cell surface CFTR internalized over time is shown. The data are expressed as the means ± S.E. ( n > 3).

Article Snippet: The cells were grown in high glucose l -glutamine Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen) and hygromycin B (150 μg/ml; Invitrogen) at 37 °C in 5% CO2 , 90% air atmosphere.

Techniques: Expressing, Incubation, Modification, Transfection, Western Blot, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Neuronatin regulates pancreatic β cell insulin content and secretion

doi: 10.1172/JCI120115

Figure Lengend Snippet: NNAT interaction with the SPC and modulation of preproinsulin handling. ( A ) Overview of affinity purification/mass spectrometry (AP/MS) screen for novel interaction partners of NNAT. Endogenous NNAT was immunoprecipitated (IP) from MIN6 cell lysates and interacting partners in co-IPs analyzed by liquid chromatography/mass spectrometry (LC/MS). ( B ) Heatmap from AP/MS analysis of top protein hits in IPs using antibodies against NNAT (NNAT Ab) and control IPs with rabbit immunoglobulins (IgG). Relatively high abundance is shown in yellow and relatively low abundance in blue. ( C ) Lysates from HEK293T cells expressing c-Myc–tagged SEC11A and FLAG-tagged NNAT were immunoprecipitated using anti–c-Myc antibodies. Proteins in input and IP samples were detected by Western blotting using anti–c-Myc and anti-FLAG antibodies. Panel shows a representative blot of 3 independent experiments. ( D ) INS1E cells transiently transfected with siRNA targeting Nnat or Sec11a were assayed in vitro for GSIS at low (3 mM) and high (25 mM) glucose. A scramble siRNA served as a control with data expressed as mean insulin secretion per unit cellular protein. Graph on the right shows total insulin content in cell lysates. ( n = 9 independent cultures per group, 3 independent experiments, 2-way ANOVA [left graph] and 1-way ANOVA [right graph].) ( E ) INS1E cells transfected with c-Myc–tagged preproinsulin and siRNAs targeting Nnat or Sec11a were pulse-labeled with 35 S-Cys/Met. Lysates immunoprecipitated with anti–c-Myc agarose were analyzed by autoradiography. Associated bar chart shows preproinsulin and proinsulin band intensities in multiple experiments quantified by densitometry and expressed as percentage processing of preproinsulin ( n = 3 cultures per group from 3 independent experiments, 1-way ANOVA). ( F ) Similar experiments performed as in E , from 3-hour chase cell lysates (C) and media (M), quantified as in E ( n = 4 cultures per group from 3 independent experiments, 1-way ANOVA for both C and M). (* P

Article Snippet: For siRNA silencing, INS1E cells ( ) (cultured in RPMI, 10% FBS, 2 mM l -glutamine, 50 μM β-mercaptoethanol, 37°C, 5% CO2 ) were transfected with 50 nM of Silencer Select siRNAs ( Nnat , s137247; Sec11a , s80747; both Ambion) using Dharmafect 1 reagent (Dharmacon).

Techniques: Affinity Purification, Mass Spectrometry, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Transfection, In Vitro, Labeling, Autoradiography

Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p

Article Snippet: Transfection medium [DMEM containing L-glutamine and 15 mm HEPES (GIBCO) supplemented with 10% NU-I serum (BD biosciences) and 0.1% selenium/insulin/transferrin (GIBCO)] was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of 1 μM ΑΤRA in serum-free medium for another 24 h. Finally, 48 hours after transfection, cells were washed with phosphate buffered saline (PBS) and then assayed for luciferase activity using the Dual Luciferase Reporter Assay system and the protocol by Promega.

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Activation Assay, Luciferase

Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: RARB and Nur77 co-operation regulate HSD3B2 in H295R cells. H295R cells were transfected with the human −1.05 kb HSD3B2 promoter constructs along with an expression vector RARB, Nur77 and in combination of RARB/Nur77. Transfection medium was changed after 6 h and cells were cultivated in either serum free or in normal growth medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA for another 24 h. ( A ) RARB and Nur77 co-operation study under serum starvation condition. ( B ) RARB and Nur77 co-operation study under normal growth condition. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of four independent experiments. * p

Article Snippet: Transfection medium [DMEM containing L-glutamine and 15 mm HEPES (GIBCO) supplemented with 10% NU-I serum (BD biosciences) and 0.1% selenium/insulin/transferrin (GIBCO)] was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of 1 μM ΑΤRA in serum-free medium for another 24 h. Finally, 48 hours after transfection, cells were washed with phosphate buffered saline (PBS) and then assayed for luciferase activity using the Dual Luciferase Reporter Assay system and the protocol by Promega.

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Activation Assay, Luciferase

Journal: Molecular Cancer

Article Title: OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway

doi: 10.1186/1476-4598-13-218

Figure Lengend Snippet: NME4 is a direct regulatory target of miR-196. (A) Differential expression of miR-196a/b and NME4 mRNA in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-qPCR. Relative levels of miR-196a/b were determined after normalization to the U6 RNA level (internal control) for each sample. The NME4 RNA level was normalized to actin for each sample. (B) Differential expression of NME4 mRNA and protein in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-PCR and western blotting. Actin expression was also determined as an internal control. (C) Inverse correlation between NME4 and miR-196a/b expression which determined by RT-qPCR. (D) Effect of miR-196 modulation on NME4 mRNA expression. OECM1 and SAS cells transfected with miR-196a/b–specific antagomirs or overexpression plasmids were harvested. Total protein was extracted and subjected to western blot analysis of NME4 expression. The relative expression was determined after normalization to actin as an internal control. (E) The mature sequences of miR-196a and miR-196b, as well as the 3′-UTR sequence of the human NME4 gene, are shown. The mutant sequence of the 3′-UTR region of NEM4 was designed. Luciferase reporter assay was performed to determine whether NME4 is a direct miR-196a/b target gene. Cells transfected with miR-196–specific antagomirs or overexpression plasmids were co-transfected with pMIR, p-UTR-WT, or p-UTR-mut. Renilla luciferase was also transfected as a reference control for each condition. Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay. (* p

Article Snippet: The immortalized normal keratinocyte cells were maintained in KSFM medium (Life Technologies, Inc., Gibco BRL, Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Sequencing, Mutagenesis, Luciferase, Reporter Assay