cftr  (Alomone Labs)

Journal: PLoS ONE

Article Title: Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

doi: 10.1371/journal.pone.0022212

Figure Lengend Snippet: Expression of CFTR in epithelial cells and monocytes. Panel A : Monocyte cell lysates were denatured at 95°C while epithelial cells were treated at 40°C before SDS-PAGE. The left panel shows the result after the incubation with the anti- CFTR antibody ACL-006, 24-1 or 13-1. Although in epithelial cells all the CFTR forms (A, B and C) are detected (although with a different pattern of reactivity depending on the type of antibody), in monocytes not all the bands are detected by the individual antibodies, although all of them are present as testified by the recognition of bands A, B and C by their combination. While band A is consistently detected by ACL-006 antibody in monocytes, detection of the other bands with this antibody is not reproducible in all the samples tested. On the right side of the panel the arrow indicates the less glycosylated form of CFTR (band A) expressed in 16HBE14o- cells, CFPAC-1 and monocytes (ACL-006 antibody). mRNA of CFTR as detected by RT-PCR is expressed by monocytes that express the same spliced isoform expressed by epithelial cells. Actin (ACTB) has been amplified as control. Panel B : Temperature-dependent detection of CFTR Effect of the denaturating temperature on the CFTR bands detected in epithelial cells and monocytes. A major band around 170 kDa (band C) is detected in both epithelial cells lines expressing wild-type CFTR (Suit 2 and 16HBE14o-) and in monocytes (13-1 antibody). CFBE41o − (CFBE) (F508del/F508del) cells express a single band corresponding to a core-glycosylated CFTR (band B, ACL-006 and 24-1 antibodies) while CFTR expression level in IB3-1 (W1282X/F508del) is below the detection limit of the assay for ACL-006 antibody but display a band using the 24-1 antibody. The incubation with ACL-006, 24-1 and 13-1 antibodies, reactive against a C- and a more N-terminal epitope (13-1), when analyzed together, identify all the glycosylated forms in our assay only when the most appropriate temperature is utilized (40°C for epithelial cells and 95°C for monocytes). Of note is the loss of CFTR signal in monocytes treated at 40°C associated to a reduction of immunoreactivity of anti-actin antibody used to demonstrate equal protein load (last two lanes, right). Panel C : Western blotting with the indicated antibodies on lysates of monocytes derived from patients with the indicated genotypes. Note that the bands corresponding to a full length CFTR present in wild-type (WT) or obligate heterozygotes (WT/Q39X) are missing in monocytes derived from patients homozygous for nonsense mutations (13-1 antibody). Patients carrying one or two F508del alleles express a much fainter band. Actin expression is shown to demonstrate equal protein load.

Article Snippet: After centrifugation at 16,000× g for 30 seconds at 4°C, the supernatant was transferred, to a new ice-cold microcentrifuge tube containing 2 µg rabbit anti-CFTR (ACL-006 Alomone Labs Ltd., Jerusalem, Israel; 0.8 mg/ml) and 2 µg mouse anti-CFTR (MAB 3482 clone MM13-4 Chemicon International, Temecula, California USA; 1 mg/ml) or non-immune rabbit and mouse IgG and incubated for 2 hours at 4°C.

Techniques: Expressing, SDS Page, Incubation, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Derivative Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Impaired Renal HCO3- Excretion in Cystic Fibrosis

doi: 10.1681/ASN.2020010053

Figure Lengend Snippet: The secretin receptor and CFTR is present in pendrin-positive cells in the cortex of WT mice. (A) Immunohistochemical localization of pendrin (red, upper panel), the SCTR (green, middle panel), and double staining (lower panel) in the cortex of WT mice. (B) Immunohistochemical localization of pendrin (red, upper panel), CFTR (green, middle panel), and double staining (lower panel) in the cortex of WT mice. (C) Higher magnification of the indicated area in (B) showing apical colocalization of CFTR and pendrin in the apical membrane. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Sections were incubated with primary antibodies against CFTR (rabbit IgG, ACL-006; Alomone Labs), SCTR (rabbit IgG; epitope, AA 170–220; bs-0089R; Bioss, Woburn, MA), and pendrin E-20 (goat IgG; Santa Cruz Biotechnology) in 0.5% BSA and 0.04% Triton X-100 overnight at 4°C, and with anti-rabbit Alexa Fluor 488 IgG or anti-goat Alexa Fluor 546 (Invitrogen) for 1 hour at 37°C.

Techniques: Mouse Assay, Immunohistochemistry, Double Staining

Journal: Life

Article Title: F1099L-CFTR (c.3297C > G) has Impaired Channel Function and Associates with Mild Disease Phenotypes in Two Pediatric Patients

doi: 10.3390/life11020131

Figure Lengend Snippet: F1099L- cystic fibrosis transmembrane conductance regulator (CFTR) has a defect in protein maturation and exhibits impairment in chloride channel function. ( A ) A representative blot showing the expression level and maturation status of F1099L-, wild type (WT)-, or F508del-CFTR, or vector transiently expressed in HEK-293 cells. Band C and B denote a mature form and an immature form of CFTR, respectively. ( B ) The total CFTR protein expression level (Band C + Band B) of F1099L-CFTR was 45% of WT-CFTR. The data were quantified from blots as represented in ( A ) using ImageJ software. The densities of the bands were normalized to their loading control, β-actin, respectively. * p

Article Snippet: Antibodies and ReagentsAntibodies: anti-CFTR (clone MM13-4, EMD Millipore Corporation, CA, USA), anti-CFTR (ACL-006, Alomone labs, Jerusalem, Israel), anti-β-actin (Sigma, MO, USA), goat anti-mouse IgG secondary antibody, HRP (Pierce Biotechnology, IL, USA), and goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, MA, USA).

Techniques: Expressing, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury

doi: 10.1038/srep15946

Figure Lengend Snippet: CFTR mediates heat-induced activation of MAPK/NF-κB/COX-2/PGE 2 in 16HBE14o- cells. ( a ) QRT-PCR analysis of CFTR and COX-2 in cells transfected with CFTR-silencing ribosome vectors (rib) or empty pEF6/V5-His vectors as negative control (pEF). ***P

Article Snippet: Antibodies used in this study were listed as follows: CFTR (1:200, Cat#ACL-006, Alomone labs, Jerusalem, Israel), COX-2 (1:200, Cat#160106, Cayman chemical, Ann Arbor, USA), P-JNK (1:1000, Cat#9255, Cell Signaling Technology, Boston, USA), JNK (1:1000, Cat#9252, Cell Signaling Technology), P-Erk1/2 (1:1000, Cat#4370, Cell Signaling Technology), Erk1/2 (1:1000, Cat#4695, Cell Signaling Technology), P-IκBα (1:500, Cat#AB55066, Sangon Biotech), GAPDH (1:5000, Cat#KC-5G5, Kangcheng Biotech, Shanghai, China), Tubulin (1:500, Cat#10759-1-AP, Proteintech, Chicago, USA) and β-actin (1:1000, Cat#60008-1-lg, Proteintech).

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Negative Control

Journal: Scientific Reports

Article Title: CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury

doi: 10.1038/srep15946

Figure Lengend Snippet: IL-8 production as a result of MAPK/NF-κB/COX-2/PGE 2 activation by heat or CFTR knockdown in 16HBE14o- cells. ( a,b ) QRT-PCR analysis and ELISA detection of IL-8 production in cells with (+) or without (−) heat-treatment ( a ), transfection of pEF/rib ( b ) in the presence (+) or absence (−) of BAY11 (40 μM), PD98059 (20 μM), SP600125 (40 μM) or DMSO as vehicle control. ### P

Article Snippet: Antibodies used in this study were listed as follows: CFTR (1:200, Cat#ACL-006, Alomone labs, Jerusalem, Israel), COX-2 (1:200, Cat#160106, Cayman chemical, Ann Arbor, USA), P-JNK (1:1000, Cat#9255, Cell Signaling Technology, Boston, USA), JNK (1:1000, Cat#9252, Cell Signaling Technology), P-Erk1/2 (1:1000, Cat#4370, Cell Signaling Technology), Erk1/2 (1:1000, Cat#4695, Cell Signaling Technology), P-IκBα (1:500, Cat#AB55066, Sangon Biotech), GAPDH (1:5000, Cat#KC-5G5, Kangcheng Biotech, Shanghai, China), Tubulin (1:500, Cat#10759-1-AP, Proteintech, Chicago, USA) and β-actin (1:1000, Cat#60008-1-lg, Proteintech).

Techniques: Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Scientific Reports

Article Title: CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury

doi: 10.1038/srep15946

Figure Lengend Snippet: Curcumin mitigates heat-induced inflammation by upregulating CFTR in airway epithelial cells in vitro and in vivo . ( a,b ) Western blotting for CFTR and COX-2 ( a ), and ELISA detection of PGE 2 and IL-8 ( b ) in 16HBE14o- cells before (−) and after (+) heat-treatment in the presence (+) or absence (−) of curcumin (Cur, 10 μM)or DMSO as vehicle control. Curcumin was administrated either 4 hours before (Pre), 0.5 hour after (Post, 0.5 h), or 8 hours after (Post, 8 h) the heat treatment. Tubulin was used as loading control. *P

Article Snippet: Antibodies used in this study were listed as follows: CFTR (1:200, Cat#ACL-006, Alomone labs, Jerusalem, Israel), COX-2 (1:200, Cat#160106, Cayman chemical, Ann Arbor, USA), P-JNK (1:1000, Cat#9255, Cell Signaling Technology, Boston, USA), JNK (1:1000, Cat#9252, Cell Signaling Technology), P-Erk1/2 (1:1000, Cat#4370, Cell Signaling Technology), Erk1/2 (1:1000, Cat#4695, Cell Signaling Technology), P-IκBα (1:500, Cat#AB55066, Sangon Biotech), GAPDH (1:5000, Cat#KC-5G5, Kangcheng Biotech, Shanghai, China), Tubulin (1:500, Cat#10759-1-AP, Proteintech, Chicago, USA) and β-actin (1:1000, Cat#60008-1-lg, Proteintech).

Techniques: In Vitro, In Vivo, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury

doi: 10.1038/srep15946

Figure Lengend Snippet: Heat-induced temporal changes in CFTR and COX-2 expression in airway epithelial cells in vitro and in vivo . ( a,b ) 16HBE14o- cells were cultured at 37 °C till 80% confluence before incubated at 52 °C for 5 min as heat-treatment and subsequently back at 37 °C for recovery. Cells were collected before (Ctrl), after heat-treatment (0 h), or after recovery for 8 hours (8 h) to 5 days (5d) for QRT-PCR ( a ) and western blot ( b ) analysis of CFTR and COX-2. Data are means ± SEM from at least three independent experiments. ***P

Article Snippet: Antibodies used in this study were listed as follows: CFTR (1:200, Cat#ACL-006, Alomone labs, Jerusalem, Israel), COX-2 (1:200, Cat#160106, Cayman chemical, Ann Arbor, USA), P-JNK (1:1000, Cat#9255, Cell Signaling Technology, Boston, USA), JNK (1:1000, Cat#9252, Cell Signaling Technology), P-Erk1/2 (1:1000, Cat#4370, Cell Signaling Technology), Erk1/2 (1:1000, Cat#4695, Cell Signaling Technology), P-IκBα (1:500, Cat#AB55066, Sangon Biotech), GAPDH (1:5000, Cat#KC-5G5, Kangcheng Biotech, Shanghai, China), Tubulin (1:500, Cat#10759-1-AP, Proteintech, Chicago, USA) and β-actin (1:1000, Cat#60008-1-lg, Proteintech).

Techniques: Expressing, In Vitro, In Vivo, Cell Culture, Incubation, Quantitative RT-PCR, Western Blot