Journal: Endocrinology

Article Title: Periovulatory expression of hydrogen peroxide-induced sulfiredoxin and peroxiredoxin 2 in the rat ovary: gonadotropin regulation and potential modification.

doi: 10.1210/en.2012-1414

Figure Lengend Snippet: FIG. 6. Regulation of ovarian PRDX2 expression by gonadotropins. A, Aliquots of total RNA (20 g) isolated from ovaries at the indicated time intervals after eCG/hCG stimulation were assayed for PRDX2 mRNA levels by Northern blotting using a rat PRDX2 cDNA probe. The expression of 28S ribosomal RNA was used as an internal standard. The PRDX2 transcript was quantified using a phosphorimager and normalized for 28S RNA levels from three independently performed experiments (mean SEM). B, In situ localization of ovarian PRDX2 mRNA. Ovarian sections were hybridized with a 35S-labeled PRDX2 cRNA probe. Photomicrographs were taken under bright-field (a, c, and e) and corresponding dark-field (b, d, and f) illumination. Arrowhead, Oocyte; arrow, theca cell; asterisk, growing follicle; P, preovulatory follicle; CL, corpus luteum, 20. C, Western blotting analysis of PRDX2 protein in the ovary. Ovarian lysates were separated on a 12% SDS-PAGE gel in the presence (reducing) or absence (nonreducing) of 10% 2-mercaptoethanol. The quantified PRDX2 protein levels are expressed as a mean SEM of two or three independently performed experiments.

Article Snippet: Blots were incubated with primary antibodies against Srx, PRDX2, -PRDX-SO2/3, p-Akt (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology), pPTEN (Cell Signaling Technology), or PTEN (Cell Signaling Technology).

Techniques: Expressing, Isolation, Northern Blot, In Situ, Labeling, Western Blot, SDS Page

Journal: Endocrinology

Article Title: Periovulatory expression of hydrogen peroxide-induced sulfiredoxin and peroxiredoxin 2 in the rat ovary: gonadotropin regulation and potential modification.

doi: 10.1210/en.2012-1414

Figure Lengend Snippet: FIG. 7. Modification of PRDX2 in preovulatory follicles after gonadotropin treatment. Preovulatory follicles were dissected from ovaries obtained at different times after eCG/hCG treatment. Cell lysates of preovulatory follicles were analyzed by SDS-PAGE (A) or two- dimensional PAGE (B) followed by immunoblot analysis with antibodies specific to Srx, sulfinic/sulfonic 2-Cys PRDXs (PRDX-SO2/3), PRDX2, and GAPDH. Lysates of HeLa cells exposed to 1 mM H2O2 for 10 min were used as a positive control for the expression of hyperoxidized 2-Cys PRDXs. The region on the two-dimensional immunoblots corresponds to a molecular mass of 22–28 kDa (vertical) and isoelectric points of 5.2–5.6 (horizontal). The band intensities of each sample were measured and the ratio of hyperoxidized (Ox) to reduced (Re) PRDX2 was used to plot the graph (B, lower panel). Each point on the graph represents mean SEM of three independently performed experiments. *, P 0.05 vs. 0 h.

Article Snippet: Blots were incubated with primary antibodies against Srx, PRDX2, -PRDX-SO2/3, p-Akt (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology), pPTEN (Cell Signaling Technology), or PTEN (Cell Signaling Technology).

Techniques: Modification, SDS Page, Western Blot, Positive Control, Expressing

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, Control, Retroviral, Plasmid Preparation, Expressing, Staining, Western Blot, FLAG-tag, Quantitative RT-PCR, Plaque Assay, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E efficiently suppresses human coronavirus spike-protein-mediated entry. (A) Levels of Ly6E, GILT, and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT, or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at 24 h after the addition of tet. The cellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). **, P < 0.001 compared to the control cells expressing CAT.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Cell Culture, Infection, Luciferase, Activity Assay, Western Blot

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and lung cancer (A549) cells. (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of cellular LY6E expression was determined by Western blotting using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E-specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). Differences in viral RNA between scramble or LY6E-specific shRNA-expressing cells were analyzed statistically (**, P < 0.001; Student’s t test). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a Western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red); cell nuclei were visualized by DAPI staining (D and F).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, shRNA, Expressing, Western Blot, Control, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Staining

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Identification of critical structural motifs essential for LY6E to restrict human coronavirus entry. (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is presented, in which the “three finger-fold” structure is highlighted with black boxes. The conserved L36 as well as the GPI anchor and N99 glycosylation sites are indicated. (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT or wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E. (D) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in medium with indicated concentrations of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (E) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E (L36A) were cultured with or without the indicated concentrations of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Sequencing, Glycoproteomics, Derivative Assay, Expressing, Control, Mutagenesis, Cell Culture, Western Blot, Infection, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits TMPRSS2-enhanced entry of human coronaviruses. Flp-In T-Rex 293-derived cell lines expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. (A) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the absence of tet. (B) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the presence of tet. (C) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation ( n = 4); **, P < 0.001 compared to cells transfected with the pCAGGS vector.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Cell Culture, Infection, Luciferase, Virus, Activity Assay, Standard Deviation

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Amphotericin B treatment compromises IFITM3 inhibition of human coronavirus entry, but have no impact on Ly6E inhibition of human coronavirus entry. Flp-In T-Rex 293-derived cell lines expressing IFITM3 (A) or LY6E (B) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1 μM AmphoB. Luciferase activity was measured at 48 h postinfection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mock treatment.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Inhibition, Derivative Assay, Expressing, Cell Culture, Infection, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Dynamic interplay between sortilin and syndecan-1 contributes to prostate cancer progression

doi: 10.1038/s41598-023-40347-7

Figure Lengend Snippet: Sortilin (SORT) interacts with progranulin (PRGN) and LPL in PCa cells and PRGN is secreted by aggressive PC3 cells. ( a ) Representative confocal images showing co-labelling of cells with anti-PRGN (red) and anti-SORT (green) antibodies. Scale bars; 10 µm. ( b ) Quantification of colocalisation between PRGN and SORT. ( c ) Amount of PRGN in conditioned media (CM) and corresponding cell lysates with quantification of band densities and ratio of PRGN in CM to PRGN in cell lysates. ( d ) Endogenous SORT was immunoprecipitated (IP) with anti-SORT antibodies and PRGN was detected by Western blotting. Input; cell lysate. ( e ) Representative confocal images showing co-labelling of cells with anti-LPL (red) and anti-SORT (green) antibodies. Scale bars; 10 µm. ( f ) Quantification of colocalisation between LPL and SORT. ( g ) Amount of LPL in CM and corresponding cell lysates with quantification of band densities in cell lysates. In ( c ) and ( g ) PNT2 bands from the same membrane were shifted towards LNCaP and PC3 bands. Western blotting signal was normalised using total protein staining. ( h ) Endogenous SORT was immunoprecipitated (IP) with anti-SORT antibodies and LPL was detected by Western blotting. Input; cell lysate. Data are presented as mean ± SD, in ( b , f ) n = 10 representative ROIs, in ( c , g ) n = 3 (independent experiments), one-way ANOVA.

Article Snippet: Human prostate cell PNT2 (ECACC 95012613), LNCaP (clone FCG, ECACC 89110211) and PC3 (ECACC 90112714) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) from CellBank Australia (Children's Medical Research Institute, Westmead, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Membrane, Staining

Journal: Cell Reports

Article Title: The α 2 δ-like Protein Cachd1 Increases N-type Calcium Currents and Cell Surface Expression and Competes with α 2 δ-1

doi: 10.1016/j.celrep.2018.10.033

Figure Lengend Snippet:

Article Snippet: For co-IPs and electrophysiological studies, tsA-201 cells were transfected using Fugene6 (Promega, Fitchburg, WI) according to the manufacturer’s protocol.

Techniques: Affinity Purification, Recombinant, Modification, Nucleic Acid Electrophoresis, Membrane, Plasmid Preparation, Bradford Assay, Mutagenesis, Software