protease inhibitor  (Roche)

Journal: Molecular psychiatry

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

doi: 10.1038/s41380-018-0027-3

Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

Journal: European Urology

Article Title: Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer

doi: 10.1016/j.eururo.2016.03.049

Figure Lengend Snippet: EP343 identifies the AR-V7 splice variant. Immunoprecipitation (IP) of M12 cells stably transfected with PCDNA3 plasmid expressing 3×FLAG-tagged cDNAs encoding either AR-FL, ARv567es, or AR-V7. Following IP of cell lysates with a FLAG antibody, blots were probed for (A) AR-NTD or (B) AR-V7 to determine the specificity of the EP343 antibody. (C) Immunoblot staining of various prostate cancer cell lines, both positive and negative for AR-V7 and AR-FL, shows AR-V7 staining (top arrow) along with a number of nonspecific bands and AR-FL (bottom arrow). (D) LNCaP95s were transfected with siRNAs targeting either cryptic exon 3B (si V7), exon 7 (si ex7), exon 1 (si ex1), scrambled SiRNA (si SCR), or an input control. Note that siRNAs directed at components of AR-V7 (si V7 and si ex 1) reduced EP343 staining, but siRNA directed at exon 7 had no effect. (E) IHC on 22Rv1 (positive for AR-NTD and AR-V7), DU145 (negative for AR-NTD and AR-V7) and PC3s which are negative for AR-NTD and should be negative for AR-V7 but have obvious EP343 staining. Both PC3 and 22Rv1 were transfected w ith AR-V7 (cryptic exon 3B) siRNA and then pelleted for EP343 IHC staining. The targeted siRNA reduced AR-V7 in 22Rv1 IHC but not in PC3.

Article Snippet: 2.1.4 Western blotting and co-immunoprecipitation Western blotting was performed on whole cell extracts prepared in lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM EGTA, 1% Triton X-100) with a complete protease inhibitor cocktail (Roche, Basel, Switzerland).

Techniques: Variant Assay, Immunoprecipitation, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Staining, Immunohistochemistry

Journal: Mediators of Inflammation

Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

doi: 10.1155/2013/271813

Figure Lengend Snippet: P2X7 activation induces cell death in EOC13 microglial cells. (a) Adherent EOC13 cells in complete DMEM medium were incubated in the absence or presence of varying concentrations of ATP (as indicated) at 37°C for 24 h. (b) Adherent cells in complete DMEM medium were preincubated in the absence or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence or presence of 2 mM ATP for 24 h. (e and f) Adherent cells in complete DMEM medium were incubated in the absence or presence of 40 mM NAC at 37°C for 90 min and incubated in the absence or presence of 2 mM ATP for the final (e) 15–60 min or (f) 45 min (of the 90 min incubation), and then the medium replaced with fresh complete DMEM medium for 24 h. (a, b, and e) Cells were harvested, labelled with Annexin-V-Fluorescein and 7AAD, and the percentage of Annexin-V − /7AAD + , Annexin-V + /7AAD − , and Annexin-V + /7AAD + cells (together representing total cell death) determined by flow cytometry. (f) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. (c) Adherent DCF-loaded cells in complete DMEM medium were incubated in the absence (basal) or presence of varying concentrations of ATP (as indicated) at 37°C for 15 min. (d) Adherent DCF-loaded cells in complete DMEM medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 2 mM ATP for 15 min. (c and d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. Results shown as (a) dot plots of one representative set of data demonstrating the quadrant markers and (a–e) means ± SD, n = 3; *** P

Article Snippet: Protease inhibitor cocktail tablets (complete, Mini, EDTA-free) and Annexin-V-Fluorescein were from Roche Diagnostics (Penzberg, Germany).

Techniques: Activation Assay, Incubation, Flow Cytometry, Cytometry, Microscopy, Centrifugation, Fluorescence