Journal: Journal of Korean Medical Science

Article Title: Immune Responses After Vaccination With Primary 2-Dose ChAdOx1 Plus a Booster of BNT162b2 or Vaccination With Primary 2-Dose BNT162b2 Plus a Booster of BNT162b2 and the Occurrence of Omicron Breakthrough Infection

doi: 10.3346/jkms.2023.38.e155

Figure Lengend Snippet: WT = wild type, SVNT = surrogate virus neutralization test, IGRA = interferon-γ release assay.

Article Snippet: Neutralizing antibody responses against wild type SARS-CoV-2 were detected using the GenScript SARS-CoV-2 cPass surrogate virus neutralization test (SVNT-WT) kit (GenScript Biotech Corporation, Piscataway, NJ, USA).

Techniques: Virus, Neutralization, Release Assay

Journal: Journal of Korean Medical Science

Article Title: Immune Responses After Vaccination With Primary 2-Dose ChAdOx1 Plus a Booster of BNT162b2 or Vaccination With Primary 2-Dose BNT162b2 Plus a Booster of BNT162b2 and the Occurrence of Omicron Breakthrough Infection

doi: 10.3346/jkms.2023.38.e155

Figure Lengend Snippet: SVNT = surrogate virus neutralization test, BNT = BNT162b2, ChAdOx = ChAdOx1 nCoV-19. To adjust the effect of the time-difference in comparing the serologic test results between the two groups, the comparison in ( A ) was not different ( P = 0.704), but that in ( B ) was different ( P = 0.001) in multiple regression models * .

Article Snippet: Neutralizing antibody responses against wild type SARS-CoV-2 were detected using the GenScript SARS-CoV-2 cPass surrogate virus neutralization test (SVNT-WT) kit (GenScript Biotech Corporation, Piscataway, NJ, USA).

Techniques: Virus, Neutralization, Comparison

Journal: Journal of Korean Medical Science

Article Title: Immune Responses After Vaccination With Primary 2-Dose ChAdOx1 Plus a Booster of BNT162b2 or Vaccination With Primary 2-Dose BNT162b2 Plus a Booster of BNT162b2 and the Occurrence of Omicron Breakthrough Infection

doi: 10.3346/jkms.2023.38.e155

Figure Lengend Snippet: SVNT = surrogate virus neutralization test, IGRA = interferon-γ release assay, BI = breakthrough infection.

Article Snippet: Neutralizing antibody responses against wild type SARS-CoV-2 were detected using the GenScript SARS-CoV-2 cPass surrogate virus neutralization test (SVNT-WT) kit (GenScript Biotech Corporation, Piscataway, NJ, USA).

Techniques: Virus, Neutralization, Release Assay, Infection

Journal: Journal of Cell Science

Article Title: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2

doi: 10.1242/jcs.173195

Figure Lengend Snippet: As a result of H 2 O 2 -induced oxidative stress the cell-cycle-stage-dependent nuclear pool of pTyr23AnxA2 is diminished and replaced by a cortical pool. PC12 cells were double-stained for immunofluorescence using mono- and polyclonal antibodies against pTyr23AnxA2 (A1,B1, green) and the S-phase marker PCNA (A2,B2, red), respectively. The insets of the merged confocal images (A3,B3) also show DAPI staining (blue) to highlight the nuclei. PC12 cells, either untreated (A1,B1,I), or treated for 15 min with 300 µM (C,D) or 1 mM (E,F,II) H 2 O 2 , were subjected to immunofluorescence staining using specific monoclonal antibodies against pTyr23AnxA2 (C–F, green) or non-phosphorylated AnxA2 (I,II, green). The insets show DNA staining by DAPI (blue). Two nuclei with different signal intensities for pTyr23AnxA2 are indicated by the arrows in A1. The arrows in B2 and B3 indicate cells in late S-phase. Scale bars: 10 µm. This key experiment was repeated more than 15 times and other experiments at least five times.

Article Snippet: Total AnxA2 or its Tyr23-phosphorylated form were detected using monoclonal antibodies directed against AnxA2 (610069, BD Biosciences, 1:1000) or pTyr23AnxA2 (sc-135753, Santa Cruz Biotechnologies, 1:200) ( ).

Techniques: Staining, Immunofluorescence, Marker

Journal: Journal of Cell Science

Article Title: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2

doi: 10.1242/jcs.173195

Figure Lengend Snippet: Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H 2 O 2 . (A) PC12 cells were grown in exosome-depleted medium in the presence of H 2 O 2 for 0 min (lanes 1,5), 15 min (lanes 2,6), 1 h (lanes 3,7), or 2 h (lanes 4,8). ECM-bound proteins were released by EGTA (lanes 1–4), whereas extracellular vesicles were isolated from the culture medium by the ExoQuick-TC method (lanes 5–8). 100 µg of protein from the EGTA-released fractions (lanes 1–4) and the control extracellular vesicle fraction (lane 5), or an equal volume of extracellular vesicles from H 2 O 2 -treated cells (lanes 6–8) were separated by 10% SDS-PAGE, transferred to nitro-cellulose membranes and probed with antibodies against pTyr23AnxA2, total AnxA2, CD63, TSG-101 and T-cadherin, as indicated. (B) Following 1 h treatment of PC12 cells with 1 mM H 2 O 2 , proteins (600 µg) present in purified extracellular vesicles were immunoprecipitated (IP) by monoclonal AnxA2 antibodies (lane 1) after pre-clearance of the samples with normal mouse IgG (lane 2). The proteins were subjected to 10% SDS-PAGE and immunoblot analysis using monoclonal antibodies against pTyr23AnxA2 or ubiquitin by loading half of the immunoprecipitation sample on each gel. The bands representing ubiquitylated AnxA2 (square bracket; Ub-AnxA2) and IgG light chain (L C , arrowhead) are indicated to the right. (A,B) Following incubation with HRP-conjugated secondary antibodies and the ECL-reagent, the reactive protein bands were detected using the ChemiDoc™ XRS+ molecular imager. Note that the secondary antibody used in B only recognises the IgG light chains. Molecular mass standards are indicated to the left. (C) PC12 cells were untreated (C1), or treated for 15 min (C2), 30 min, (C3), 60 min (C4) or 120 min (C5) with 1 mM H 2 O 2 . The localisation of pTyr23AnxA2, detected using the monoclonal antibody (green), is shown in the merged images, which also display nuclear staining (DAPI, blue). Scale bars: 10 µm.

Article Snippet: Total AnxA2 or its Tyr23-phosphorylated form were detected using monoclonal antibodies directed against AnxA2 (610069, BD Biosciences, 1:1000) or pTyr23AnxA2 (sc-135753, Santa Cruz Biotechnologies, 1:200) ( ).

Techniques: Isolation, SDS Page, Purification, Immunoprecipitation, Western Blot, Incubation, Staining

Journal: Journal of Cell Science

Article Title: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2

doi: 10.1242/jcs.173195

Figure Lengend Snippet: Cortical AnxA2 is phosphorylated by the Src kinase. (A–F) Cortical Tyr23 phosphorylation of AnxA2 is blocked by the Src kinase inhibitor PP2. PC12 cells were incubated for 5 h either in control medium (A), or medium containing 20 µM PP2 (B,E) or PP3 (C,F), after which 1 mM H 2 O 2 was added to the control (D), PP2- (E) and PP3-pre-treated (F) cells for 15 min. The panels show staining of the cells with the antibody against pTyr23AnxA2 (green), whereas the insets represent merged images showing also nuclear staining by DAPI (blue). Scale bars: 10 µm. (G) pTyr23AnxA2 is mainly present as high-molecular-mass forms in the nucleus. Nuclear and cytoplasmic fractions were prepared from control and PP2-treated cells, as indicated. 150 µg of the proteins in each fraction were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes for western blot analysis with monoclonal antibodies against pTyr23AnxA2 or AnxA2 with loading controls for the nucleus (fibrillarin) or cytoplasm (tubulin), as indicated. (H) Src kinase in the cytoplasm is activated during the 15 min treatment with 1 mM H 2 O 2 . Nuclear and cytoplasmic fractions were prepared from control and H 2 O 2 -treated cells, as indicated. 100 µg of the proteins in each fraction were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes for western blot analysis employing antibodies against activated (pTyr416 Src is a marker of Src activation) and total Src. The loading controls for the nucleus (fibrillarin) and cytoplasm (tubulin) are also indicated. (I) Distribution of marker proteins in sub-cellular fractions. 100 μg of proteins from the cytoplasmic (Cyt) or nuclear (Nu) fractions derived from PC12 cells were separated by 10% (w/v) SDS-PAGE and transferred to a nitrocellulose membrane, which was cut in two parts prior to western blot analysis employing antibodies against GAPDH (lower part) or topoisomerase (upper part). Detection of the resulting protein bands was performed using the ChemiDoc™ XRS+ molecular imager after incubation with HRP-complex-conjugated secondary antibodies and ECL-reagent. The arrows to the left of G indicate the protein molecular mass standards.

Article Snippet: Total AnxA2 or its Tyr23-phosphorylated form were detected using monoclonal antibodies directed against AnxA2 (610069, BD Biosciences, 1:1000) or pTyr23AnxA2 (sc-135753, Santa Cruz Biotechnologies, 1:200) ( ).

Techniques: Incubation, Staining, SDS Page, Western Blot, Marker, Activation Assay, Derivative Assay

Journal: Journal of Cell Science

Article Title: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2

doi: 10.1242/jcs.173195

Figure Lengend Snippet: Nuclear pTyr23AnxA2 is found in PML bodies, but absent in nuclear speckles, whereas AnxA2 is found in SC-35-positive nuclear speckles. PC12 cells were untreated (A1–A3,B1–B3,C1–C3), or treated for 1 h with 3 μg/ml AcD (D1–D3). The cells were double-stained with monoclonal pTyr23AnxA2 (green) (A1,C1,D1) and polyclonal Fox3 (A2) or PML (C2,D2) (red) antibodies as indicated. Other cells were double-stained with polyclonal AnxA2 (B1, green) and monoclonal SC-35 (B2, red) antibodies. The merged confocal images (insets in A3,B3,C3,D3) also show DAPI staining (blue) of the nuclei. Scale bar: 10 µm. (A4,B4,C4,D4) Fluorescence intensity profiles for the two markers indicated of the cross-sections (from left to right) of control (A–C) and AcD-treated (D) cells indicated by lines on the corresponding images. Arrows in B3 indicate colocalisation of AnxA2 with SC-35-positive nuclear speckles.

Article Snippet: Total AnxA2 or its Tyr23-phosphorylated form were detected using monoclonal antibodies directed against AnxA2 (610069, BD Biosciences, 1:1000) or pTyr23AnxA2 (sc-135753, Santa Cruz Biotechnologies, 1:200) ( ).

Techniques: Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2

doi: 10.1242/jcs.173195

Figure Lengend Snippet: Nuclear export of AnxA2, but not of pTyr23AnxA2, is blocked by LmB. PC12 cells were untreated (A,C) or treated for 2 h with LmB (B,D), followed by immunofluorescence staining using monoclonal pTyr23AnxA2 (A,B) or polyclonal AnxA2 (C,D) antibodies. The middle panels show higher magnification images with lines indicating cross-sections of nuclei, with the left-to-right orientation of the lines corresponding to the fluorescence intensity profiles shown to the right. The insets include DAPI staining (blue) to verify the position of the nuclei. Scale bars: 10 µm. (E) A schematic model of the transient Tyr23 AnxA2 phosphorylation events occurring at two distinct sites in response to H 2 O 2 . Upon exposure to H 2 O 2 , nuclear Tyr23-phosphorylated AnxA2 in PML bodies rapidly dephosphorylates and might become associated with SC-35-positive nuclear speckles. AcD increases the localisation of pTyr23AnxA2 in PML bodies suggesting a role in transcription. Note that the pTyr23AnxA2 form is not exported from the nucleus (via the CRM1-dependent pathway). At the same time cortical AnxA2 is Tyr23 phosphorylated, associates with actin and subsequently with endosomes and multivesicular bodies (MVB) and is exported out of the cell in the lumen of exosomes. pTyr23AnxA2 appears to be ubiquitylated and/or sumoylated.

Article Snippet: Total AnxA2 or its Tyr23-phosphorylated form were detected using monoclonal antibodies directed against AnxA2 (610069, BD Biosciences, 1:1000) or pTyr23AnxA2 (sc-135753, Santa Cruz Biotechnologies, 1:200) ( ).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Cancer Medicine

Article Title: Exosome‐mediated delivery of miR‐204‐5p inhibits tumor growth and chemoresistance

doi: 10.1002/cam4.3248

Figure Lengend Snippet: Characteristics of exosomes derived from miR‐204‐5p‐overexpressing HEK293T cells. A, The markers of exosomes (CD63 and Flotillin‐2) were detected in HEK293T cells and exosomes by Western blot. B, The transmission electron micrograph showed roundshaped vesicles with bilayered membranes ranging from 100 nm to 150 nm in diameter released by HEK293T cells. Scale bar = 200 nm. C, 293T EXOs size distribution was measured by Zetasizer. D, Real‐time qRT‐PCR revealed that the level of miR‐204‐5p was higher in 293T‐204 cells and miR‐204 EXO than miR‐204‐3p. *** P < .001. Shown are mean ± SEM from three independent experiments

Article Snippet: The protein samples of cells and exosomes were detected with antibodies against CD63 (1:1000; BOSTER), Flotillin‐2 (1:500; Santa Cruz), RAB22A (1:1000; Proteintech), Bcl2 (1:1000; Santa Cruz) and β‐actin (1:2000; Thermo) as we previously described.,

Techniques: Derivative Assay, Western Blot, Transmission Assay, Quantitative RT-PCR

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Placement of VEGF-A mRNA injections in relation to the microdialysis probe in the rabbit study. For further details, see methods.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Placement of VEGF-A mRNA injections in relation to the microdialysis probe in the pig study. For further details, see methods.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Concentrations of human VEGF-A in eluates from 100 kDa microdialysis probes intradermally inserted in the rabbit hind leg. Microdialysis was started at t=0 h and four id injections of VEGF-A mRNA injections (50 μ g each) were given at t=1 h. Dotted line indicates Lower Limit of Quantification (LLOQ, 33.4 pg/mL). Two probes were inserted in each rabbit. Values shown are mean ± SEM (n=4).

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Concentrations of human VEGF-A in microdialysis eluates from 100 kDa microdialysis probes intradermally inserted on the pig (n=3) abdomen. Values presented as individual values in eluate from each probe. Six intradermal VEGF-A mRNA injections (50 μ L each, total 24, 120, or 600 μ g VEGF-A mRNA) were given at t=0 h. At t=1.5 h after the injections, microdialysis was started. Eluates were collected during the time intervals from 1.5 h to 3.5 h, from 3.5 h to 5.5 h, and from 5.5 h to 7.5 h, respectively. Dotted vertical line indicates Lower Limit of Quantification (33.4 pg/mL); eluate samples below LLOQ are depicted as 0.5 ∗ LLOQ=16.7 pg/mL. Six probes were inserted in each pig, two probes per dose except the 120 μ g dose, where only four probes were used to assess human VEGF-A production following injection of 24 or 600 μ g VEGF-A mRNA. The remaining two probes were used to study effects following injection of citrate/saline vehicle.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques: Injection, Saline

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Amounts (pg/mg) of human VEGF-A in skin biopsies excised approximately 8 hours after intradermal injection of VEGF-A mRNA in 3 pigs. Each dose was administered as 6 (24 and 600 μ g dose) or 4 (120 μ g dose) separate injections at two different sites in each pig. Hatched data point in the 24 μ g dose, depicted at 0.1 pg/mg, indicating amount below Lower Limit of Quantification (0.28 pg/mg).

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques: Injection

Journal: Redox Biology

Article Title: Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis

doi: 10.1016/j.redox.2017.08.015

Figure Lengend Snippet: Altered iron homeostatic mechanisms drive senescent cells to acquire intracellular iron. (A) Expression of key iron homeostasis proteins was analysed in senescent MEFs (IR) at 21 days post-irradiation (10 Gy) by western blot and densitometry. The levels of transferrin receptor 1 (TfR1) [main iron (Fe 3+ ) importer], divalent metal transporter 1 (DMT1) (cytosolic iron importer), ferroportin (iron exporter) and ferritin (intracellular iron storage) were measured in comparison to primary (PRI) MEFs. β -actin was detected as a loading control. (B) Western blot analyses and densitometry confirm elevated expression of ferritin in ( i ) senescent human diploid fibroblasts (HDF IR) and in (ii) senescent human prostate epithelial cells (PrEC IR), both at 21 days post-irradiation (10 Gy). Furthermore, elevated ferritin expression was confirmed in ( iii ) replicative senescent MEFs (P7) cultured for 21 days. β -actin was detected as loading controls. (C) Expression of key regulatory proteins of iron homeostasis was analysed in senescent MEFs (IR) at day 21 post-irradiation (10 Gy) by western blot and densitometry. The levels of iron regulatory protein 1 (IRP1) and iron-sulfur cluster assembly enzyme (ISCU) were found significantly lower than those in primary (PRI) MEFs, while IRP2 was significantly elevated. β -actin was detected as a loading control. Statistical analysis was performed by student- t -test: significant (* p < 0.05, ** p < 0.01, *** p < 0.001). Data represented as mean ± SD ( n = 3).

Article Snippet: Ferroportin was detected with primary anti-ferroportin antibody (Cat#NBP1-21502, Novus Biologicals) and secondary goat anti-rabbit IgG Alexa 488.

Techniques: Expressing, Irradiation, Western Blot, Comparison, Control, Cell Culture