Journal: PLoS ONE

Article Title: Down-Regulation of Canonical and Up-Regulation of Non-Canonical Wnt Signalling in the Carcinogenic Process of Squamous Cell Lung Carcinoma

doi: 10.1371/journal.pone.0057393

Figure Lengend Snippet: Immunofluorescent staining of SAEC. B : Immunofluorescent staining of normal A549. C : Immunofluorescent staining of Wnt11 overexpressing A549. D : Immunofluorescent staining of H157 monolayer cell cultures. (60x image, red: β-catenin, blue: DAPI). Note the dramatic increase in nuclear localization and the decrease in cellular membrane localization of A549 AC, Wnt11-A549 and H157 SCC cell lines compared to the normal pulmonary epithelium (SAEC). Data presented are representative of three independent experiments. E : Densitometry of immunofluorescent images of SAEC, A549, Wnt-11-A549 and H157 cells. Note the increased nuclear localization of β-catenin particularly in the Wnt11-A549 cell line. (M: cellular membrane, CS: cytosol, N: nucleus).

Article Snippet: Murine anti-β-catenin IgG 1 mAb (Santa Cruz) (1∶50) and donkey anti-murine IgG secondary antibody conjugated to NorthernLight 557 (R&D Systems) (1∶200) were used for immunofluorescent labelling; nuclei were counterstained with DAPI.

Techniques: Staining, Membrane

Journal: Virology

Article Title: Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus

doi: 10.1016/j.virol.2019.08.030

Figure Lengend Snippet: Determining the response of AK-D 2.2 cells to type I interferon and characterizing the replication kinetics and plaque formation of FIPV Black in AK-D 2.2 cells. A) AK-D (black) or AK-D 2.2 (green) cells were treated with increasing concentrations of feline interferon alpha (IFNα). After 6 h, total RNA was extracted and analyzed by qPCR for ISG54 and feline β -actin. ISG54 mRNA expression was normalized to β -actin then presented as average 2 −ΔCt values. B) Growth kinetics of FIPV Black (MOI = 0.1) determined by plaque assay of infected AK-D (black) and AK-D 2.2 (green) cell supernatants. Data representative of two to three independent experiments performed in triplicate and presented mean ± SD. Values were analyzed by unpaired t -tests. ***P < 0.001. C) Fcwf-4 CU cells were infected with MOI = 0.1 serotype I FIPV Black and supernatants were collected at 24 HPI after which plaque assay analysis was performed on AK-D, AK-D 2.2, or Fcwf-4 CU indicator cells. Images were taken at 48 (top) and 72 (bottom) HPI. Images are representative of three independent experiments.

Article Snippet: Membranes were blocked at 4 °C overnight in 5% milk in TBST followed by 90 min room temperature incubation in primary antibodies specific for TMPRSS2(V5) tag (murine anti-V5 monoclonal IgG; Fisher Scientific #R96025) or β -actin (murine anti-actin monoclonal IgG; Genscript #A00702-40) diluted 1:2500 and 1:5000, respectively, in 5% milk-TBST.

Techniques: Expressing, Plaque Assay, Infection

Journal: Virology

Article Title: Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus

doi: 10.1016/j.virol.2019.08.030

Figure Lengend Snippet: Evaluating the response to type I interferon and the kinetics of FIPV Black replication in Fcwf-4 parent and IFNαR-deficient cells. A) Fcwf-4 CU, Fcwf-4 CU 2.2 Poly, Fcwf-4 CU 2.2 clones 1, 2 and 3 (IRN cells) were treated with 0 or 1000 U feline IFNα for 6 h and ISG54 transcripts were measured by qPCR. B) FIPV Black growth kinetics (MOI = 0.1) at indicated hours post-infection (HPI) in Fcwf-4 CU and IRN cells. C) Nucleocapsid gene transcript levels measured by qPCR during FIPV Black infection (MOI = 0.1) of Fcwf-4 CU or IRN cells. D) FIPV Black growth kinetics (MOI = 0.01) in indicated cells pre-treated with 0 or 1000 U feline IFNα for 8 h. E) Plaque formation induced by FIPV Black in Fcwf-4 CU or IRN cells at 3–4 days post-infection (DPI). Wells display 10 − 5 virus dilution. F) Plaque sizes (mm 2 ) measured from FIPV Black plaque assays (6-well plates) at 3 and 4 DPI on Fcwf-4 CU and IRN indicator cells at 10 − 5 and 10 − 6 virus dilutions. For mRNA expression, Ct values were normalized to β -actin using the 2 −ΔCt method and presented as fold expression over mock (A) or relative expression (B). Data represent 3 independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. Viral titers (B and D) were calculated from triplicate plaque assays per time point on Fcwf-4 CU indicator cells and represent 3 independent experiments. Mean ± SD plaque-forming units (PFU) per mL analyzed by two-way ANOVA by time (D). Plaque sizes (F) were measured using Adobe Photoshop software and mean ± SD values were analyzed using unpaired t-tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Membranes were blocked at 4 °C overnight in 5% milk in TBST followed by 90 min room temperature incubation in primary antibodies specific for TMPRSS2(V5) tag (murine anti-V5 monoclonal IgG; Fisher Scientific #R96025) or β -actin (murine anti-actin monoclonal IgG; Genscript #A00702-40) diluted 1:2500 and 1:5000, respectively, in 5% milk-TBST.

Techniques: Clone Assay, Infection, Virus, Expressing, Software

Journal: Virology

Article Title: Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus

doi: 10.1016/j.virol.2019.08.030

Figure Lengend Snippet: Fcwf-4 IRN cells express an mRNA predicted to generate a truncated, null-mutant IFNαR protein. A) Deduced nucleotide and amino acid sequences of the IFNαR2 region targeted by Crispr/Cas technology. Single-guide RNA sequence target (underlined); protospacer-adjacent motif (PAM) (yellow); STOP codon and asterisk (red) indicate early termination of translation of the IFNαR2 protein in IRN cells. B) Feline IFNαR2 exon 1 expression determined from total RNAs collected from Fcwf-4 CU and Fcwf-4 IRN confluent monolayers. mRNA expression normalized to β -actin and presented as average 2 −ΔCt expression values. Data represent two independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. **P < 0.01. C) Melt curves and maximum melt temperatures of IFNαR2 amplicons produced during qPCR of RNAs obtained from Fcwf-4 CU or Fcwf-4 IRN cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were blocked at 4 °C overnight in 5% milk in TBST followed by 90 min room temperature incubation in primary antibodies specific for TMPRSS2(V5) tag (murine anti-V5 monoclonal IgG; Fisher Scientific #R96025) or β -actin (murine anti-actin monoclonal IgG; Genscript #A00702-40) diluted 1:2500 and 1:5000, respectively, in 5% milk-TBST.

Techniques: Mutagenesis, CRISPR, Sequencing, Expressing, Produced

Journal: Virology

Article Title: Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus

doi: 10.1016/j.virol.2019.08.030

Figure Lengend Snippet: Expressing TMPRSS2 in feline cells and evaluating the effect on FIPV Black replication. A) Immunofluorescence detection of feline TMPRSS2(V5) in HEK 293T/17 cells. 200 ng of pLVX-fTMPRSS2(V5) or pLVX (mock) was transfected into HEK 293T/17 cells for 18 h. Cells were stained with mouse-anti-V5 (1:500), and 1:1000 Alexa Flor 568-conjugated goat-anti-mouse IgG was used to visualize TMPRSS2(V5); Hoesch 3342 (1:1000) was used to stain nuclei. B) Detection of feline TMPRSS2(V5) by Western blot following transfection of HEK 293T/17 cells (left) or transduction of Fcwf-4 IRN cells with pLVX lentiviruses encoding TMPRSS2(V5) (right). The full length (55 kDa) and cleavage product (>25 kDa) of TMPRSS2 are indicated. Feline β -actin used to visualize protein loading. C-D) The impact of feline TMPRSS2 expression on FIPV replication evaluated in Fcwf-4 IRN cells. Indicated dilutions of pLVX-fTMPRSS2(V5) or Mock (empty) transducing particles were applied to Fcwf-4 IRN cells for 48 h prior to infection with FIPV Black (MOI = 0.1). RNA was isolated after 18 h infection and qPCRs were performed to detect TMPRSS2 (C) , N gene (D) , and β -actin transcripts. mRNA expression normalized to β -actin and presented as average 2 −ΔCt expression values. Data represent two independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. **P < 0.01; ***P < 0.001; not significant (ns).

Article Snippet: Membranes were blocked at 4 °C overnight in 5% milk in TBST followed by 90 min room temperature incubation in primary antibodies specific for TMPRSS2(V5) tag (murine anti-V5 monoclonal IgG; Fisher Scientific #R96025) or β -actin (murine anti-actin monoclonal IgG; Genscript #A00702-40) diluted 1:2500 and 1:5000, respectively, in 5% milk-TBST.

Techniques: Expressing, Immunofluorescence, Transfection, Staining, Western Blot, Transduction, Infection, Isolation

Journal: Journal of Virology

Article Title: A novel replication-deficient FCV vaccine provides strong immune protection in cats

doi: 10.1128/jvi.00093-25

Figure Lengend Snippet: Rescue and characterization of rHBDL2 FCV-△VP2. ( a ) Schematic representation illustrating the construction of infectious clones of rHBDL2 FCV-△VP2, featuring a targeted deletion of nucleotides 19–48 at the N-terminus of the FCV VP2 gene. ( b ) rHBDL2 FCV-△VP2 was successfully rescued in a cell line stably expressing the VP2 protein (F81-VP2 cells). ( c ) Conventional F81 cells were infected with either rHBDL2 FCV-△VP2 or wild-type HBDL2 FCV at an MOI of 0.001, and Western blot analysis was conducted to assess the VP1 protein expression levels at 6, 12, 18, and 24 h post-infection, with β-actin used as an internal control. ( d ) The viral titers (TCID 50 ) were assessed at 4, 6, 12, 24, 36, 48, 60, and 72 h post-infection to construct a growth curve for p5 and p10 rHBDL2 FCV-△VP2, as well as WT HBDL2 FCV (MOI = 0.001), in F81-VP2 cells. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparison test, with significance levels denoted as follows: * P < 0.05, ** P < 0.01. The color-coded asterisk (*) and underline (_) denote comparisons between viral strains.

Article Snippet: After being blocked with 5% skim milk, the membranes were incubated overnight at 4°C with laboratory-preserved murine anti-VP1 monoclonal antibody (dilution 1:2,000) and murine anti-β-actin monoclonal antibody (dilution 1:2,000; Abbkine, Wuhan, China).

Techniques: Clone Assay, Stable Transfection, Expressing, Infection, Western Blot, Control, Construct, Comparison

Journal: Journal of Virology

Article Title: A novel replication-deficient FCV vaccine provides strong immune protection in cats

doi: 10.1128/jvi.00093-25

Figure Lengend Snippet: N-terminal deletion of the FCV VP2 protein inhibited viral genome replication without impeding virion assembly. ( a ) Conventional F81 cells were infected with either rHBDL2 FCV-△VP2 or WT HBDL2 FCV at an MOI of 50. Western blot analysis was conducted to assess the levels of the VP1 protein in the cell supernatant, the cell lysate supernatant, and within the cells at 6 h post-infection, with β-actin used as an internal control. Quantitative analysis of the gray values was performed to measure the bands corresponding to the VP1 protein in the cell lysate supernatant ( b ) and within the cells ( c ). Additionally, RT-qPCR analysis was employed to determine the relative levels of the FCV genome within the cells ( d ). ( e ) Transmission electron microscopy was used to observe the morphology of the inc.rHBDL2 FCV-△VP2, rHBDL2 FCV-△VP2, and WT HBDL2 FCV viral particles, as indicated by black arrows. ( f ) The relative genomic levels of FCV in conventional F81 cells infected with inc.rHBDL2 FCV-△VP2 and WT HBDL2 FCV were quantified via RT‒qPCR at 0, 0.5, 1.0, 1.5, 4.0, and 6.0 h. Statistical analysis was conducted via unpaired t -tests, with significance denoted as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: After being blocked with 5% skim milk, the membranes were incubated overnight at 4°C with laboratory-preserved murine anti-VP1 monoclonal antibody (dilution 1:2,000) and murine anti-β-actin monoclonal antibody (dilution 1:2,000; Abbkine, Wuhan, China).

Techniques: Infection, Western Blot, Control, Quantitative RT-PCR, Transmission Assay, Electron Microscopy

Journal: Oncotarget

Article Title: Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

doi:

Figure Lengend Snippet: In vitro deglycosylation of CDCP1 employing Neuraminidase (A) Endo H (B) and PNGase F (C) . Hydrolyzed lysates from PC3, N2, and ML2 cells were separated on SDS-PAGE and immunoblotted with anti-CDCP1 (CS4115). In vivo inhibition of glycosylation of CDCP1 in which PC3, N2, and ML2 cells were treated with tunicamycin (D) or swainsonine (E) in vivo for 24 h. The total cell lysate was extracted, subjected to SDS-PAGE and immunoblotted with anti-CDCP1 (CS4115). β-actin was used as a loading control. Shown are HMW-CDCP1 and LMW-CDCP1. (F) Sialylation of HMW-CDCP1 protein was quantified by metabolically labeling sialyl proteins with ManNAz followed by immunoprecipitation of normalized amounts of CDCP1 with anti-CDCP1 (CS4115). A click reaction was performed to label the azido-sugar with biotin to allow for subsequent blotting with IRDye 800-conjugated streptavidin. (G) Normalized amounts of HMW-CDCP1 from N2 and ML2 cell was immunoprecipitated with anti-CDCP1 (CS4115) subjected to SDS-PAGE and immunoblotted with linkage-specific lectins SNA, MALII, and WGA as indicated.

Article Snippet: Murine monoclonal anti-β-actin was from BD PharMingen (San Diego, CA) and rabbit polyclonal anti-GAPDH was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, SDS Page, In Vivo, Inhibition, Metabolic Labelling, Labeling, Immunoprecipitation

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: ENaC expression in kidney lysates prepared at different temperatures. Kidney homogenate was prepared in Laemmli buffer with beta‐mercaptoethanol either at room temperature (RT) for 10 min, 37°C for 30 min, or 97°C for 3 or 10 min (denoted for each lane). Three separate replicates of these conditions were run and the subsequent membranes were probed with Stressmarq antibodies directed against each ENaC subunit: (a) α, (b) β, or (c) γ. Asterisks denote the expected α‐ and β‐subunits on their respective membranes. The γ‐subunit displays both a full‐length band and a cleaved product. Blots are representative of results from at least three separate validations.

Article Snippet: In our hands, we found that anti‐β‐ and γ‐subunit antibodies from a commonly used commercial source, StressMarq, work well in identifying ENaC subunit expression with both immunoblotting and immunofluorescence imaging in murine tissues.

Techniques: Expressing

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Mouse kidney, but not lung, demonstrate differences in ENaC α‐ and γ‐subunit expression with aldosterone. Lysate from mouse kidney and lung collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were blotted for the presence of each ENaC subunit. (a) The Stressmarq anti‐α‐subunit antibody revealed an intense 80 kDa nonspecific band, denoted by ** and a 95 kDa full length α‐subunit, denoted by *. The top panel was exposed for a longer period (~7 min) to show the 95 kDa band and the bottom panel shows a quick initial exposure (~5 s) before saturation of the 80 kDa band occurred. (b) The blot was stripped and reprobed with a previously characterized antibody, produced in the Loffing laboratory. The panel shows two exposures, separated by a dashed line, to reveal both the full‐length 95 kDa α‐subunit, denoted by *, and a 30 kDa α‐subunit N‐terminal cleavage product. (c) The Stressmarq antibody directed against the β‐subunit shows the presence of a band at 90 kDa (denoted by *). (d) The Stressmarq antibody directed against the γ‐subunit revealed bands corresponding to a full‐length 80 kDa γ‐subunit and 70 kDa cleavage products, as indicated. (e) Signal from the kidney samples was quantified by densitometry, with each band normalized to total protein. Quantification is shown as a fold change from the average HS signal with p values shown for relationships that were significant ( p < 0.05) as assessed by multiple t ‐tests. (f) Mouse lung lysate was first probed with the Stressmarq α antibody, followed by a light chain only secondary antibody (LC‐only HRP). The blot was then stripped and reprobed again with the Stressmarq α antibody but followed by a whole IgG secondary antibody (H + L HRP). The blot was then stripped again and reprobed with the Loffing α antibody, followed by a whole IgG secondary antibody (H + L HRP). The Stressmarq antibody revealed an intense nonspecific band of ~80 kDa (denoted by **) and a full length α‐subunit migrating ~95 kDa (denoted by *). The Loffing antibody reveal both the full‐length 95 kDa α‐subunit and a 30 kDa N‐terminal α‐subunit cleavage product. Blots are representative of results from at least three separate experiments.

Article Snippet: In our hands, we found that anti‐β‐ and γ‐subunit antibodies from a commonly used commercial source, StressMarq, work well in identifying ENaC subunit expression with both immunoblotting and immunofluorescence imaging in murine tissues.

Techniques: Expressing, Produced

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Subunit‐specific antibodies demonstrate linearity across a wide range of protein concentrations. Decreasing amounts of lung or kidney homogenate were probed for each subunit to determine the working range of the antibody. (a) Lung lysate ranging from 80 to 2.5 μg total protein, as denoted along the top of the blot, was probed for the α‐subunit using the StressMarq antibody. (b) The band of interest, denoted by *, was quantified and normalized to the value obtained for 40 μg (the halfway value) so that the results from three separate replicates could be combined. The dashed line demonstrates perfect linearity. (c) Kidney lysate was utilized for the β‐subunit and (e) the γ‐subunit. (d, f) The quantification of each band was again performed as described for (b), with both the full‐length and cleavage product bands being quantified for the γ‐subunit. Each graph represents results obtained from three separate experiments.

Article Snippet: In our hands, we found that anti‐β‐ and γ‐subunit antibodies from a commonly used commercial source, StressMarq, work well in identifying ENaC subunit expression with both immunoblotting and immunofluorescence imaging in murine tissues.

Techniques:

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Immunofluorescent staining reveals movement of the β‐ and γ‐subunits from an intracellular location to apical surface with a high K + diet. Kidneys from mice kept on a control diet (top) or 4 days of a high K + diet (bottom) were probed for expression of (a) the β‐subunit of ENaC or (b) the γ‐subunit of ENaC. Sections were counterstained for AQP2 (green) to indicate principal cells. The basolateral surfaces of tubules are denoted by solid lines and the apical surface by dashed lines. Scale bars represent 20 μm and images are representative of three separate regions examined in three mice of each treatment.

Article Snippet: In our hands, we found that anti‐β‐ and γ‐subunit antibodies from a commonly used commercial source, StressMarq, work well in identifying ENaC subunit expression with both immunoblotting and immunofluorescence imaging in murine tissues.

Techniques: Staining, Expressing