Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Bronchial Secretory Immunoglobulin A Deficiency Correlates With Airway Inflammation and Progression of Chronic Obstructive Pulmonary Disease

doi: 10.1164/rccm.201010-1629oc

Figure Lengend Snippet: Figure 7. Polymeric immunoglobulin re- ceptor (pIgR) expression and IgA transcytosis in air-liquid interface (ALI) cultured human bronchial epithelial cells (HBECs) grown with or without retinoic acid (RA). (A) Pseu- dostratified ciliated structure of bronchial epithelium and marked secretory compo- nent (SC)/pIgR expression in culture with RA added; squamous stratified structure of bronchial epithelium and absent SC/pIgR expression in culture grown without RA. Top row: hematoxylin and eosin (HE)–stained par- affin sections (original magnification 3400). Bottom row: immunohistochemistry of par- affin tissue sections with goat polyclonal anti–SC-pIgR antibody (original magnifica- tion 3400). (B) Western blot showing ex- pression of SC/pIgR in cell cultures with RA added and no pIgR expression in cells grown without RA. (C) PIGR mRNA expres- sion in RA1 and RA2 cultures (normalized to HPRT). (D) IgA and secretory IgA (SIgA) concentrations in apical washings of ALI cultured HBECs grown with or without RA. * P , 0.001 (compared with RA1 cul- tures). Mean 6 SEM is indicated for each concentration.

Article Snippet: Goat polyclonal antiSC/pIgR antibody (R&D Systems, Minneapolis, MN) was used for IHC and Western blotting.

Techniques: Expressing, Cell Culture, Staining, Immunohistochemistry, Western Blot, Concentration Assay

Journal: Nature medicine

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy

doi: 10.1038/nm.2181

Figure Lengend Snippet: CIB1 facilitates calcineurin/NFAT activation. ( a ) Neonatal rat cardiac myocytes were infected with AdNFAT-luc and Adβgal, AdCIB1 or AdCain and stimulated with PE (10 μM) for 48 hrs as indicated. NFAT-dependent luciferase activity was measured and normalized to total protein content. N=6–15 per condition. *p<0.001 vs. Adβgal and Adβgal + PE, #p<0.001 vs. AdCIB1 + PE. ( b ) Neonatal rat cardiac myocytes were infected with AdNFAT-luc and Adβgal, AdCIB1 or AdCIB1Δ and stimulated with PE for 48 hrs as indicated. N=5–15 per condition, *p<0.001 vs. Adβgal and Adβgal + PE, #p<0.001 vs. AdCIB1 + PE. ( c ) Calcineurin phosphatase assay over 30 minutes against the phosphorylated RII peptide with recombinant calcineurin holoenzyme (2.5 μg = 20 units) and CIB1 (5 μg). ( d ) Neonatal rat cardiac myocytes were infected with AdNFAT-luc and transfected with control or CIB1 siRNA, then stimulated with PE for 24 hrs as indicated. N=6 per condition, *p<0.001 vs. control siRNA, #p<0.001 vs. control siRNA + PE. ( e ) NFAT luciferase activity (normalized to total protein content) measured from NFAT-luc reporter transgenic mice WT or null (−/−) for Cib1 after sham and TAC surgery as indicated. N=3–6 mice per condition, *p<0.05 vs. WT sham. ( f ) Immunoblot for the indicated proteins from WT or Ppp3r1 fl/fl mouse embryonic fibroblasts (MEFs) infected with Adβgal control or AdCre to delete CnB1 protein if appropriate. ( g ) Immunoblot for the α1C subunit of the L-type Ca 2+ channel and CIB1 after IP from CIB1 overexpressing mouse hearts 2 weeks after TAC or sham operation. CIB1 antibody or control IgG was used for the IP. ( h ) Immunoblot against the α1C subunit of the L-type Ca 2+ channel after a pull-down assay from neonatal cardiomyocyte lysate incubated with the indicated GST-CIB1 fusion constructs. ( i ) Neonatal rat cardiac myocytes were transfected with control or CIB1 siRNA and infected with AdNFAT-luc, then stimulated with PE and verapamil (10μM) for 24 hours as indicated. N=4–6 per condition, *p<0.05 vs. control siRNA, #p<0.001 vs. control siRNA + PE.

Article Snippet: Western blot analysis was performed using the following antibodies: phospo-Akt (Serine 473), Akt, phospho-GSK3β (Serine 9), GSK3β, phospho-ERK1/2, ERK 1/2, phospho-JNK, JNK, phospho-p38, p38, Myc-Tag (Cell Signaling Technology), CIB1 (Zymed Laboratories Inc.), Pan-CnA, atrial natriuretic factor (Chemicon International), CnB (Upstate), pan-cadherin and Na + /K + ATPase (Abcam), β1-intergrin, GST, IκB-α, SERCA2 (Santa Cruz Biotechnology Inc.), α1C subunit of the L-type Ca 2+ channel (Alomone labs) and GAPDH (Fitzgerald Industries Intl).

Techniques: Activation Assay, Infection, Luciferase, Activity Assay, Phosphatase Assay, Recombinant, Transfection, Transgenic Assay, Western Blot, Pull Down Assay, Incubation, Construct