Journal: F1000Research

Article Title: An unexpected effect of TNF-α on F508del-CFTR maturation and function

doi: 10.12688/f1000research.6683.2

Figure Lengend Snippet: Differentiated primary HBE cell cultures grown at an air-liquid interface were incubated with 50ng/ml TNFα for 10–30min, 3–6h and 24h. CFTR immunodetection was performed with 24.1 anti-CFTR antibody and analyzed with confocal microscopy. Green staining represents CFTR (Alexa Fluor 488), red color staining represents ZO-1 protein of tight junctions (Alexa Fluor 594) and blue DAPI staining visualizes nuclei. Independent TNFα treatments and CFTR immunodetection were performed on HBE cells from three different F508del/F508del CF patients. Representative images of one experiment are demonstrated (Scale bars = 20µm).

Article Snippet: Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031).

Techniques: Incubation, Immunodetection, Confocal Microscopy, Staining

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 1. Western blot analysis of CFTR and STAT1 expression in un- treated (lane 1), PMA-treated (lane 2), TNF-treated (lane 3), and IFN- treated (lane 4) T84 (A) and RCMC (B). T84 cells were treated with PMA (10 ng/ml), human recombinant TNF (10 ng/ml), or IFN (10 ng/ml) for 24 h. RCMC were treated with PMA (10 ng/ml) or rat recombinant TNF (10 ng/ml) or IFN (10 ng/ml) for 24 h. Cell lysates were resolved on an 4 to 12% SDS-PAGE and blotted with anti-CFTR, anti-STAT1, or anti-actin antibody. Western blots shown represent three independent experiments. The effect of 24 h IFN treatment (80 ng/ml) on CFTR expression was characterized in T84 cells (C) and rat peritoneal mast cells (D), using confocal laser scanning microscopy (bar on the figure represents 10 m).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Western Blot, Expressing, Recombinant, SDS Page, Confocal Laser Scanning Microscopy

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 4. A, IFN-mediated up-regulation of mast cell CFTR protein requires activation of JAK2 and/or p38 and ERK. RCMC, LAD2, and T84 were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SB202190; 30 mg/ml), or ERK MAPK inhibitor (U0126; 30 mg/ml) for 24 h. Representative of three inde- pendent experiments. B, densitometry summary of data in Fig. 4. RCMC were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SP202190; 30 mg/ml), or ERK inhibitor (U0126; 30 mg/ml) for 24 h, and expression of CFTR (black columns) and STAT1 (gray columns) was analyzed by Western blot (n 3 independent experiments; error bars represent S.E.M.). As- terisks represent statistical significance as determined by Student’s t test (p 0.05).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Oncology Letters

Article Title: Overexpression of close homolog of L1 enhances the chemosensitivity of lung cancer cells via inhibition of the Akt pathway

doi: 10.3892/ol.2020.11972

Figure Lengend Snippet: CHL1 is downregulated in DDP and PTX-resistant A549 cells. (A) Cell survival of A549 and A549-resistant cells (A549/DDP and A549/PTX) treated with increasing concentrations of DDP and PTX, as assessed by MTT assay. (B) The IC 50 values of DDP in A549/DDP and A549 cells, and the IC 50 values of PTX in A549/PTX and A549 cells. *P<0.05 vs. A549 cells. (C) Western blotting demonstrated the expression of drug resistance-related proteins MDR1, MRP and LRP in A549 cells and A549-resistant cells (A549/DDP and A549/PTX). *P<0.05 vs. A549 cells. The protein and mRNA expression levels of CHL1 in A549 cells and A549-resistant cells (A549/DDP and A549/PTX) were analysed by (D) western blotting and (E) reverse transcription-quantitative PCR, respectively. *P<0.05 vs. A549 cells. (F) The mRNA expression of CHL1 in H460 and H460/DDP cells in the GSE21656 dataset. *P<0.05 vs. H460 cells. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel; MDR1, multi-drug resistance gene 1; MRP, multidrug resistance-associated protein; LRP, low-density lipoprotein receptor-related protein; IC50, half maximal inhibitory concentration.

Article Snippet: Next, the proteins were transferred onto a polyvinylidene membrane (Thermo Fisher Scientific, Inc.), blocked with 5% BSA (Thermo Fisher Scientific, Inc.) for 2 h at 4°C, and incubated overnight at 4°C with primary antibodies against CHL1 (1:500; cat. no. 25250-1-AP; ProteinTech, Inc.), multi-drug resistance gene 1 (MDR1; 1:500; cat. no. 22336-1-AP; ProteinTech, Inc.), multidrug resistance-associated protein (MRP; 1:500; cat. no. 67228-1-Ig; ProteinTech, Inc.), low-density lipoprotein receptor-related protein (LRP; 1:500; cat. no. 22336-1-AP; ProteinTech, Inc.), phosphorylated (p)-Akt (1:1,000; cat. no. ab38449; Abcam,) and Akt (1:2,000; cat. no. ab227385; Abcam).

Techniques: MTT Assay, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Concentration Assay