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blocking buffer  (Rockland Immunochemicals)


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    Structured Review

    Rockland Immunochemicals blocking buffer
    Blocking Buffer, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/blocking+solution/Blocking+Buffer+Sampler+Kit/pm25771199-93-12-18
    Average 93 stars, based on 28 article reviews
    blocking buffer - by Bioz Stars, 2026-11
    93/100 stars

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    Related Articles

    Membrane:

    Article Title: Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors.
    Article Snippet: SDS-PAGE was performed using NuPAGE 4–12 % Bis-Tris gradient gel and a compatible MES SDS buffer system (both from Thermo Fisher Scientific). .. Resolved proteins were electrotransferred to a nitrocellulose membrane, blocked with fluorescent Western blocking buffer (Rockland Immunochemicals), and probed with designated primary antibodies. .. After three washes in PBS containing 0.05 % (v/v) Tween-20, the nitrocellulose membranes were probed with Alexa Fluor-680 conjugated secondary antibody (Thermo Fisher Scientific).

    Article Title: Combining Fundamental Kinetics and Standard Alkylation Assays to Prioritize Lead-Like KRAS G12C Inhibitors
    Article Snippet: Plates were shaken for 30 min at 500 rpm at 4oC, mixed three times with a multichannel pipette and spun at 2000 rpm for 15 min. Lysate from each well (20 μl) was added to 10 μl of 4x loading buffer (Invitrogen) and 3 μl of 10x reducing agent (Invitrogen) and then boiled for 10 min at 95° C. 15 μl of reduced/boiled lysate was loaded on a 4-20 %, 15-well TRIS-Glycine gel and run for 165 min and 110V and then transferred to a nitrocellulose membrane using iBlot Gell Tranfer Stacks (Invitrogen). .. The membrane was blocked for 1 h with blocking buffer at room temp (Rockland), and then incubated at 4°C overnight with KRAS antibody (Proteintech 12063-1-AP) diluted 1:1000 in blocking buffer. .. The next day, after several washes in TBST (Sigma), the membrane was stained with fluorescent anti-rabbit antibody (LI-COR 926-32211) for 1 h at room KRAS migrates around 21K while alkylated KRAS has a slightly higher molecular weight that results in a visible electrophoretic shift.

    Western Blot:

    Article Title: Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors.
    Article Snippet: SDS-PAGE was performed using NuPAGE 4–12 % Bis-Tris gradient gel and a compatible MES SDS buffer system (both from Thermo Fisher Scientific). .. Resolved proteins were electrotransferred to a nitrocellulose membrane, blocked with fluorescent Western blocking buffer (Rockland Immunochemicals), and probed with designated primary antibodies. .. After three washes in PBS containing 0.05 % (v/v) Tween-20, the nitrocellulose membranes were probed with Alexa Fluor-680 conjugated secondary antibody (Thermo Fisher Scientific).

    Article Title: Distinct Filament Conformation for Receptor-Bound Amyloid-ß from Alzheimer’s Disease Brain
    Article Snippet: The resulting samples were separated on 4-20% gradient Criterion Tris-Glycine gels (BioRad), transferred onto the nitrocellulose membranes, blocked with Rockland fluorescent Western Blot blocking buffer and immunoprobed with the appropriate detection antibodies overnight at 4C, washed in PBST, incubated the IRdye conjugated secondary antibodies (LiCor) for 1 h at RT and imaged on the near-infrared scanner (LiCor Odyssey). .. Both primary and secondary antibody were diluted in Rockland fluorescent Western Blot blocking buffer. .. The immunoblot data quantitation was performed using the LiCor ImageStudio software.

    Article Title: Distinct Filament Conformation for Receptor-Bound Amyloid-ß from Alzheimer’s Disease Brain
    Article Snippet: .. The resulting samples were separated on 4-20% gradient Criterion Tris-Glycine gels (BioRad), transferred onto the nitrocellulose membranes, blocked with Rockland fluorescent Western Blot blocking buffer and immunoprobed with the appropriate detection antibodies overnight at 4C, washed in PBST, incubated the IRdye conjugated secondary antibodies (LiCor) for 1 h at RT and imaged on the near-infrared scanner (LiCor Odyssey). .. Both primary and secondary antibody were diluted in Rockland fluorescent Western Blot blocking buffer.

    Article Title: Methods and compositions for the treatment of amyloid-related disorders
    Article Snippet: Following the BN-PAGE, the gels were transferred onto PVDF membranes using iBlot semi-dry transfer (ThermoFisher), the membranes were dried and the excess of Coomassie dye was removed by washing the membrane in methanol three times for 5 min to avoid interference with subsequent immunoblotting. .. The membranes were then washed 3× with water, blocked using fluorescent western blot blocking buffer (Rockland) and immunoblotted with 1:500 SAF-32 mouse anti-PrPC antibody (Cayman Chemical) in TBS-T followed by 800CW-conjugated donkey anti-mouse secondary antibody (LiCor). ..

    Article Title: Neuronal transcriptome, tau and synapse loss in Alzheimer’s knock-in mice require prion protein
    Article Snippet: .. Nitrocellulose membranes were blocked in blocking buffer for fluorescent western blotting (Rockland; MB-070–010) for 1 h at room temperature and incubated overnight in primary antibodies at 4 °C. .. Anti-pTau S202/ T205 (AT8) (Thermo Fisher Scientific #MN1020, 1:700), anti-C1Q (Dako A0136; 1:1000), anti-beta Actin (D6A8) Rabbit mAb (Cell Singnaling 8457S; 1:2000) and anti-beta Actin (Abcam ab8226; 1:5000) were used as primaries in this experiment.

    Blocking Assay:

    Article Title: Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors.
    Article Snippet: SDS-PAGE was performed using NuPAGE 4–12 % Bis-Tris gradient gel and a compatible MES SDS buffer system (both from Thermo Fisher Scientific). .. Resolved proteins were electrotransferred to a nitrocellulose membrane, blocked with fluorescent Western blocking buffer (Rockland Immunochemicals), and probed with designated primary antibodies. .. After three washes in PBS containing 0.05 % (v/v) Tween-20, the nitrocellulose membranes were probed with Alexa Fluor-680 conjugated secondary antibody (Thermo Fisher Scientific).

    Article Title: Distinct Filament Conformation for Receptor-Bound Amyloid-ß from Alzheimer’s Disease Brain
    Article Snippet: The resulting samples were separated on 4-20% gradient Criterion Tris-Glycine gels (BioRad), transferred onto the nitrocellulose membranes, blocked with Rockland fluorescent Western Blot blocking buffer and immunoprobed with the appropriate detection antibodies overnight at 4C, washed in PBST, incubated the IRdye conjugated secondary antibodies (LiCor) for 1 h at RT and imaged on the near-infrared scanner (LiCor Odyssey). .. Both primary and secondary antibody were diluted in Rockland fluorescent Western Blot blocking buffer. .. The immunoblot data quantitation was performed using the LiCor ImageStudio software.

    Article Title: Distinct Filament Conformation for Receptor-Bound Amyloid-ß from Alzheimer’s Disease Brain
    Article Snippet: .. The resulting samples were separated on 4-20% gradient Criterion Tris-Glycine gels (BioRad), transferred onto the nitrocellulose membranes, blocked with Rockland fluorescent Western Blot blocking buffer and immunoprobed with the appropriate detection antibodies overnight at 4C, washed in PBST, incubated the IRdye conjugated secondary antibodies (LiCor) for 1 h at RT and imaged on the near-infrared scanner (LiCor Odyssey). .. Both primary and secondary antibody were diluted in Rockland fluorescent Western Blot blocking buffer.

    Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance
    Article Snippet: .. Nonspecific binding was blocked by incubation with blocking buffer (Rockland) for 60 min at room temperature. .. The membranes were probed for ASS1 [Cell Signaling Technology (CST), 70720], GAPDH (CST, 2118S), ERα (Abcam, ab32063), HER2 (CST, 2242) and Phosphor-CAD (Ser1859) (CST,1266).

    Article Title: Patient Brain Organoids Identify a Link between the 16p11.2 Copy Number Variant and the RBFOX1 Gene.
    Article Snippet: .. The samples were quenched with 0.1 M glycine at 7.4 pH for 20 min, followed by 30 min incubation in a blocking buffer: 5% goat serum (Rockland, D104000050) or 5% normal donkey serum (Millipore Sigma, 566460), 0.1% TritonX-100 in 1× PBS. ..

    Article Title: Methods and compositions for the treatment of amyloid-related disorders
    Article Snippet: Following the BN-PAGE, the gels were transferred onto PVDF membranes using iBlot semi-dry transfer (ThermoFisher), the membranes were dried and the excess of Coomassie dye was removed by washing the membrane in methanol three times for 5 min to avoid interference with subsequent immunoblotting. .. The membranes were then washed 3× with water, blocked using fluorescent western blot blocking buffer (Rockland) and immunoblotted with 1:500 SAF-32 mouse anti-PrPC antibody (Cayman Chemical) in TBS-T followed by 800CW-conjugated donkey anti-mouse secondary antibody (LiCor). ..

    Article Title: Neuronal transcriptome, tau and synapse loss in Alzheimer’s knock-in mice require prion protein
    Article Snippet: .. Nitrocellulose membranes were blocked in blocking buffer for fluorescent western blotting (Rockland; MB-070–010) for 1 h at room temperature and incubated overnight in primary antibodies at 4 °C. .. Anti-pTau S202/ T205 (AT8) (Thermo Fisher Scientific #MN1020, 1:700), anti-C1Q (Dako A0136; 1:1000), anti-beta Actin (D6A8) Rabbit mAb (Cell Singnaling 8457S; 1:2000) and anti-beta Actin (Abcam ab8226; 1:5000) were used as primaries in this experiment.

    Article Title: Combining Fundamental Kinetics and Standard Alkylation Assays to Prioritize Lead-Like KRAS G12C Inhibitors
    Article Snippet: Plates were shaken for 30 min at 500 rpm at 4oC, mixed three times with a multichannel pipette and spun at 2000 rpm for 15 min. Lysate from each well (20 μl) was added to 10 μl of 4x loading buffer (Invitrogen) and 3 μl of 10x reducing agent (Invitrogen) and then boiled for 10 min at 95° C. 15 μl of reduced/boiled lysate was loaded on a 4-20 %, 15-well TRIS-Glycine gel and run for 165 min and 110V and then transferred to a nitrocellulose membrane using iBlot Gell Tranfer Stacks (Invitrogen). .. The membrane was blocked for 1 h with blocking buffer at room temp (Rockland), and then incubated at 4°C overnight with KRAS antibody (Proteintech 12063-1-AP) diluted 1:1000 in blocking buffer. .. The next day, after several washes in TBST (Sigma), the membrane was stained with fluorescent anti-rabbit antibody (LI-COR 926-32211) for 1 h at room KRAS migrates around 21K while alkylated KRAS has a slightly higher molecular weight that results in a visible electrophoretic shift.

    Incubation:

    Article Title: Distinct Filament Conformation for Receptor-Bound Amyloid-ß from Alzheimer’s Disease Brain
    Article Snippet: .. The resulting samples were separated on 4-20% gradient Criterion Tris-Glycine gels (BioRad), transferred onto the nitrocellulose membranes, blocked with Rockland fluorescent Western Blot blocking buffer and immunoprobed with the appropriate detection antibodies overnight at 4C, washed in PBST, incubated the IRdye conjugated secondary antibodies (LiCor) for 1 h at RT and imaged on the near-infrared scanner (LiCor Odyssey). .. Both primary and secondary antibody were diluted in Rockland fluorescent Western Blot blocking buffer.

    Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance
    Article Snippet: .. Nonspecific binding was blocked by incubation with blocking buffer (Rockland) for 60 min at room temperature. .. The membranes were probed for ASS1 [Cell Signaling Technology (CST), 70720], GAPDH (CST, 2118S), ERα (Abcam, ab32063), HER2 (CST, 2242) and Phosphor-CAD (Ser1859) (CST,1266).

    Article Title: Patient Brain Organoids Identify a Link between the 16p11.2 Copy Number Variant and the RBFOX1 Gene.
    Article Snippet: .. The samples were quenched with 0.1 M glycine at 7.4 pH for 20 min, followed by 30 min incubation in a blocking buffer: 5% goat serum (Rockland, D104000050) or 5% normal donkey serum (Millipore Sigma, 566460), 0.1% TritonX-100 in 1× PBS. ..

    Article Title: Neuronal transcriptome, tau and synapse loss in Alzheimer’s knock-in mice require prion protein
    Article Snippet: .. Nitrocellulose membranes were blocked in blocking buffer for fluorescent western blotting (Rockland; MB-070–010) for 1 h at room temperature and incubated overnight in primary antibodies at 4 °C. .. Anti-pTau S202/ T205 (AT8) (Thermo Fisher Scientific #MN1020, 1:700), anti-C1Q (Dako A0136; 1:1000), anti-beta Actin (D6A8) Rabbit mAb (Cell Singnaling 8457S; 1:2000) and anti-beta Actin (Abcam ab8226; 1:5000) were used as primaries in this experiment.

    Article Title: Combining Fundamental Kinetics and Standard Alkylation Assays to Prioritize Lead-Like KRAS G12C Inhibitors
    Article Snippet: Plates were shaken for 30 min at 500 rpm at 4oC, mixed three times with a multichannel pipette and spun at 2000 rpm for 15 min. Lysate from each well (20 μl) was added to 10 μl of 4x loading buffer (Invitrogen) and 3 μl of 10x reducing agent (Invitrogen) and then boiled for 10 min at 95° C. 15 μl of reduced/boiled lysate was loaded on a 4-20 %, 15-well TRIS-Glycine gel and run for 165 min and 110V and then transferred to a nitrocellulose membrane using iBlot Gell Tranfer Stacks (Invitrogen). .. The membrane was blocked for 1 h with blocking buffer at room temp (Rockland), and then incubated at 4°C overnight with KRAS antibody (Proteintech 12063-1-AP) diluted 1:1000 in blocking buffer. .. The next day, after several washes in TBST (Sigma), the membrane was stained with fluorescent anti-rabbit antibody (LI-COR 926-32211) for 1 h at room KRAS migrates around 21K while alkylated KRAS has a slightly higher molecular weight that results in a visible electrophoretic shift.

    Binding Assay:

    Article Title: Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance
    Article Snippet: .. Nonspecific binding was blocked by incubation with blocking buffer (Rockland) for 60 min at room temperature. .. The membranes were probed for ASS1 [Cell Signaling Technology (CST), 70720], GAPDH (CST, 2118S), ERα (Abcam, ab32063), HER2 (CST, 2242) and Phosphor-CAD (Ser1859) (CST,1266).



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    Image Search Results


    Expression and localization of CA2 in the human placentas and Serum. (A) Heatmap of carbonic anhydrase family expression in placental transcriptomes from normal and preeclamptic pregnancies. (B) Box plot analysis of carbonic anhydrase family expression in placental transcriptomes from normal and preeclamptic pregnancies. (C) MRNA expression level of CA2 in PE and normal placentas detected by RT‐qPCR in the discovery cohort. (D) Representative western blot bands for CA2. (E) Protein expression level of CA2 in PE and normal placentas detected by Western Blot in the discovery cohort. (F) Serum expression level of CA2 in PE and normal women detected by ELISA in the serum validation cohort. (G) Immunohistochemistry method to detect the expression localization of CA2 in placentas from normal and preeclamptic pregnancies. Scale bars: 3 mm (overview), 200 μm (magnified field). All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: Expression and localization of CA2 in the human placentas and Serum. (A) Heatmap of carbonic anhydrase family expression in placental transcriptomes from normal and preeclamptic pregnancies. (B) Box plot analysis of carbonic anhydrase family expression in placental transcriptomes from normal and preeclamptic pregnancies. (C) MRNA expression level of CA2 in PE and normal placentas detected by RT‐qPCR in the discovery cohort. (D) Representative western blot bands for CA2. (E) Protein expression level of CA2 in PE and normal placentas detected by Western Blot in the discovery cohort. (F) Serum expression level of CA2 in PE and normal women detected by ELISA in the serum validation cohort. (G) Immunohistochemistry method to detect the expression localization of CA2 in placentas from normal and preeclamptic pregnancies. Scale bars: 3 mm (overview), 200 μm (magnified field). All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Expressing, Analysis, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

    Correlation analysis between CA2 expression and clinical indicators. (A) Correlation of CA2 levels with SBP. (B) Correlation of CA2 levels with DBP. (C) Correlation of CA2 levels with Fetal Weight. (D) Correlation of CA2 levels with APGAR‐1 min. (E) Correlation of CA2 levels with APGAR‐5 min. (F) Correlation of CA2 levels with Proteinuria. (G) Correlation of CA2 levels with maternal blood creatinine levels before delivery. (H) Correlation of CA2 levels with blood urea levels. (I) Correlation of CA2 levels with ALT. (J) Correlation of CA2 levels with AST. (K) Correlation of CA2 levels with PT levels. (L) Correlation of CA2 levels with APTT. (M) Correlation of CA2 levels with platelet levels. Correlation analyses were performed using Pearson's correlation coefficient. ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: Correlation analysis between CA2 expression and clinical indicators. (A) Correlation of CA2 levels with SBP. (B) Correlation of CA2 levels with DBP. (C) Correlation of CA2 levels with Fetal Weight. (D) Correlation of CA2 levels with APGAR‐1 min. (E) Correlation of CA2 levels with APGAR‐5 min. (F) Correlation of CA2 levels with Proteinuria. (G) Correlation of CA2 levels with maternal blood creatinine levels before delivery. (H) Correlation of CA2 levels with blood urea levels. (I) Correlation of CA2 levels with ALT. (J) Correlation of CA2 levels with AST. (K) Correlation of CA2 levels with PT levels. (L) Correlation of CA2 levels with APTT. (M) Correlation of CA2 levels with platelet levels. Correlation analyses were performed using Pearson's correlation coefficient. ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Analysis, Expressing

    Preeclampsia modeling and placental CA2 expression in L‐NAME mouse model. (A) Experimental workflow of the L‐NAME‐induced preeclampsia model. (B) Changes in systolic blood pressure in L‐NAME and CONTROL groups. (C, E) Body weights of fetal mice in L‐NAME and CONTROL groups. (D) Placental efficiency of fetal mice in L‐NAME and CONTROL groups. (H) SFLT‐1 levels of mice in L‐NAME and CONTROL groups. (F) The ratio of urinary albumin and creatine of mice in L‐NAME and CONTROL groups. (G, I) Representative renal and placental stained with hematoxylin & eosin (H&E) in L‐NAME and CONTROL groups. Scale bars: 250 μm. (J) Ratio of palcental labyrinth and junction zone of mice in L‐NAME and CONTROL groups. (K) MRNA expression level of placental CA2 in L‐NAME and CONTROL groups detected by RT‐qPCR. (L) Representative western blot bands for CA2. (M) Protein expression level of placental CA2 in L‐NAME and CONTROL groups detected by Western Blot. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: Preeclampsia modeling and placental CA2 expression in L‐NAME mouse model. (A) Experimental workflow of the L‐NAME‐induced preeclampsia model. (B) Changes in systolic blood pressure in L‐NAME and CONTROL groups. (C, E) Body weights of fetal mice in L‐NAME and CONTROL groups. (D) Placental efficiency of fetal mice in L‐NAME and CONTROL groups. (H) SFLT‐1 levels of mice in L‐NAME and CONTROL groups. (F) The ratio of urinary albumin and creatine of mice in L‐NAME and CONTROL groups. (G, I) Representative renal and placental stained with hematoxylin & eosin (H&E) in L‐NAME and CONTROL groups. Scale bars: 250 μm. (J) Ratio of palcental labyrinth and junction zone of mice in L‐NAME and CONTROL groups. (K) MRNA expression level of placental CA2 in L‐NAME and CONTROL groups detected by RT‐qPCR. (L) Representative western blot bands for CA2. (M) Protein expression level of placental CA2 in L‐NAME and CONTROL groups detected by Western Blot. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Expressing, Control, Staining, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Effects of CA2 on trophoblast cell function. (A) RT‐qPCR analysis to detect the expression of mRNA level after overexpression of CA2 in HTR‐8/SVneo cells. (B) Representative western blot bands for CA2. (C) Western Blot analysis to detect the expression of protein level after overexpression of CA2 in HTR‐8/SVneo cells. (D) Effect of CA2 overexpression on the proliferative capacity of HTR‐8/SVneo cells. (E) Impact of CA2 overexpression on the migration and invasion abilities of HTR‐8/SVneo cells. (F) Statistical analysis of the Transwell migration and invasion assays. (G) Effect of CA2 overexpression on the apoptotic properties of HTR‐8/SVneo cells. (H) Statistical analysis of the apoptosis assay. pEX‐3‐control: Empty vector control group; pEX‐3‐CA2: CA2 overexpression group. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: Effects of CA2 on trophoblast cell function. (A) RT‐qPCR analysis to detect the expression of mRNA level after overexpression of CA2 in HTR‐8/SVneo cells. (B) Representative western blot bands for CA2. (C) Western Blot analysis to detect the expression of protein level after overexpression of CA2 in HTR‐8/SVneo cells. (D) Effect of CA2 overexpression on the proliferative capacity of HTR‐8/SVneo cells. (E) Impact of CA2 overexpression on the migration and invasion abilities of HTR‐8/SVneo cells. (F) Statistical analysis of the Transwell migration and invasion assays. (G) Effect of CA2 overexpression on the apoptotic properties of HTR‐8/SVneo cells. (H) Statistical analysis of the apoptosis assay. pEX‐3‐control: Empty vector control group; pEX‐3‐CA2: CA2 overexpression group. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Cell Function Assay, Quantitative RT-PCR, Analysis, Expressing, Over Expression, Western Blot, Migration, Apoptosis Assay, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    VEGFA overexpression rescues the impaired proliferation, migration and invasion of HTR‐8/SVneo cells induced by CA2 overexpression. (A) Heatmap showing hierarchical clustering of differentially expressed genes between the control and CA2 overexpression groups. (B) Volcano plot of differentially expressed genes. (C) Western Blot analysis of CA2 and VEGFA to detect the protein expression level on the of HTR‐8/SVneo cells in each expremental group. (D) Proliferative capacity analysis of HTR‐8/SVneo cells in the control group、CA2 overexpression group and CA2 and VEGFA co‐overexpression group. (E) Migration and invasion abilities of HTR‐228/SVneo cells in the control group, CA2 overexpression group and CA2 and VEGFA co‐overexpression group. (F) Statistical analysis of the Transwell migration and invasion abilities in the control group, CA2 overexpression group and CA2 and VEGFA co‐overexpression group. pEX‐3‐control 1/2/3: Empty vector control group; pEX‐3‐CA2 1/2/3: CA2 overexpression group; pEX‐3‐control: Empty vector control group; pEX‐3‐ CA2: CA2 overexpression group; pEX‐3‐ CA2+ pEX‐3‐ VEGFA: CA2 and VEGFA co‐overexpression group. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: VEGFA overexpression rescues the impaired proliferation, migration and invasion of HTR‐8/SVneo cells induced by CA2 overexpression. (A) Heatmap showing hierarchical clustering of differentially expressed genes between the control and CA2 overexpression groups. (B) Volcano plot of differentially expressed genes. (C) Western Blot analysis of CA2 and VEGFA to detect the protein expression level on the of HTR‐8/SVneo cells in each expremental group. (D) Proliferative capacity analysis of HTR‐8/SVneo cells in the control group、CA2 overexpression group and CA2 and VEGFA co‐overexpression group. (E) Migration and invasion abilities of HTR‐228/SVneo cells in the control group, CA2 overexpression group and CA2 and VEGFA co‐overexpression group. (F) Statistical analysis of the Transwell migration and invasion abilities in the control group, CA2 overexpression group and CA2 and VEGFA co‐overexpression group. pEX‐3‐control 1/2/3: Empty vector control group; pEX‐3‐CA2 1/2/3: CA2 overexpression group; pEX‐3‐control: Empty vector control group; pEX‐3‐ CA2: CA2 overexpression group; pEX‐3‐ CA2+ pEX‐3‐ VEGFA: CA2 and VEGFA co‐overexpression group. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Over Expression, Migration, Control, Western Blot, Analysis, Expressing, Plasmid Preparation

    HIF‐1α activates the transcription of CA2. (A, B) RT‐qPCR analysis of CA2 and HIF‐1α to detect the expression of mRNA level under hypoxic or normoxia exposure in HTR‐8/Svneo cells. (C) Representative western blot bands. (D, E) Western Blot analysis of CA2 and HIF‐1α to detect the expression of protein level under hypoxic or normoxia exposure in HTR‐8/Svneo cells. (F, G) RT‐qPCR analysis of CA2 and HIF‐1α to detect the expression of mRNA level after overexpression HIF‐1α in HTR‐8/Svneo cells. (H) Representative western blot bands. (I, J) Western Blot analysis of CA2 and HIF‐1α to detect the expression of protein level after overexpression HIF‐1α in HTR‐8/Svneo cells. (K) Luciferase activity of HTR‐8/SVneo cells with or without overexpression HIF‐1α. (L) Luciferase activity of HTR‐8/SVneo cells with or without exposing hypoxic environment. pcDNA3.1‐control: Empty vector control group; pcDNA3.1‐HIF‐1α: HIF‐1α overexpression group; CA2 promoter + Vecter: Plasmid with CA2 promoter + empty vector control group; CA2 promoter + HIF‐1α OE: Plasmid with CA2 promoter + HIF‐1α overexpression group; CA2 promoter + Vecter + hypoxia 12 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 12 h; CA2 promoter + Vecter + hypoxia 24 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 24 h; CA2 promoter + Vecter + hypoxia 36 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 36 h. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: HIF‐1α activates the transcription of CA2. (A, B) RT‐qPCR analysis of CA2 and HIF‐1α to detect the expression of mRNA level under hypoxic or normoxia exposure in HTR‐8/Svneo cells. (C) Representative western blot bands. (D, E) Western Blot analysis of CA2 and HIF‐1α to detect the expression of protein level under hypoxic or normoxia exposure in HTR‐8/Svneo cells. (F, G) RT‐qPCR analysis of CA2 and HIF‐1α to detect the expression of mRNA level after overexpression HIF‐1α in HTR‐8/Svneo cells. (H) Representative western blot bands. (I, J) Western Blot analysis of CA2 and HIF‐1α to detect the expression of protein level after overexpression HIF‐1α in HTR‐8/Svneo cells. (K) Luciferase activity of HTR‐8/SVneo cells with or without overexpression HIF‐1α. (L) Luciferase activity of HTR‐8/SVneo cells with or without exposing hypoxic environment. pcDNA3.1‐control: Empty vector control group; pcDNA3.1‐HIF‐1α: HIF‐1α overexpression group; CA2 promoter + Vecter: Plasmid with CA2 promoter + empty vector control group; CA2 promoter + HIF‐1α OE: Plasmid with CA2 promoter + HIF‐1α overexpression group; CA2 promoter + Vecter + hypoxia 12 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 12 h; CA2 promoter + Vecter + hypoxia 24 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 24 h; CA2 promoter + Vecter + hypoxia 36 h: Plasmid with CA2 promoter + empty vector control group + exposing hypoxic environment 36 h. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Quantitative RT-PCR, Analysis, Expressing, Western Blot, Over Expression, Luciferase, Activity Assay, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    Inhibition of CA2 partially improves outcomes in a L‐NAME‐induced PE mouse model. (A) Experimental workflow for LNP‐NC and LNP‐siCA2 treatment in L‐NAME PE mice. (B) MRNA expression level of placental CA2 in LNP‐NC and LNP‐siCA2 groups detected by RT‐qPCR. (C) Representative immunofluorescence staining of CA2 in LNP‐NC and LNP‐siCA2 groups. Scale bars: 500 μm. (D) Quantitative analysis of mean fluorescence intensity of CA2 immunofluorescence signals n LNP‐NC and LNP‐siCA2 groups. (E) Changes in systolic blood pressure in LNP‐NC and LNP‐siCA2 groups. (F) The ratio of urinary albumin and creatine of mice in LNP‐NC group and LNP‐siCA2 group. (G) Representative renal stained with hematoxylin & eosin (H&E) in LNP‐NC and LNP‐siCA2 groups. (H) SFLT‐1 levels of mice in LNP‐NC and LNP‐siCA2 groups. (I, K) Body weights of fetal mice in LNP‐NC and LNP‐siCA2 groups. (J) Placental efficiency of fetal mice in LNP‐NC group and LNP‐siCA2 group. (L) Representative placental stained with hematoxylin & eosin (H&E) in LNP‐NC and LNP‐siCA2 groups. Scale bars: 250 μm. (M) Ratio of palcental labyrinth and junction zone of mice in LNP‐NC and LNP‐siCA2 groups. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: The FASEB Journal

    Article Title: Hypoxia‐Driven CA2 Promotes Trophoblast Dysfunction in Preeclampsia

    doi: 10.1096/fj.202602024R

    Figure Lengend Snippet: Inhibition of CA2 partially improves outcomes in a L‐NAME‐induced PE mouse model. (A) Experimental workflow for LNP‐NC and LNP‐siCA2 treatment in L‐NAME PE mice. (B) MRNA expression level of placental CA2 in LNP‐NC and LNP‐siCA2 groups detected by RT‐qPCR. (C) Representative immunofluorescence staining of CA2 in LNP‐NC and LNP‐siCA2 groups. Scale bars: 500 μm. (D) Quantitative analysis of mean fluorescence intensity of CA2 immunofluorescence signals n LNP‐NC and LNP‐siCA2 groups. (E) Changes in systolic blood pressure in LNP‐NC and LNP‐siCA2 groups. (F) The ratio of urinary albumin and creatine of mice in LNP‐NC group and LNP‐siCA2 group. (G) Representative renal stained with hematoxylin & eosin (H&E) in LNP‐NC and LNP‐siCA2 groups. (H) SFLT‐1 levels of mice in LNP‐NC and LNP‐siCA2 groups. (I, K) Body weights of fetal mice in LNP‐NC and LNP‐siCA2 groups. (J) Placental efficiency of fetal mice in LNP‐NC group and LNP‐siCA2 group. (L) Representative placental stained with hematoxylin & eosin (H&E) in LNP‐NC and LNP‐siCA2 groups. Scale bars: 250 μm. (M) Ratio of palcental labyrinth and junction zone of mice in LNP‐NC and LNP‐siCA2 groups. All quantitative data are expressed as means ± SD; ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: After PBS washing, nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody incubation: Excess blocking solution was discarded, and sections were incubated with primary antibody targeting CA2 (Cat. 16961‐AP, Proteintech, Wuhan, China) overnight at 4°C in a humidified dark box.

    Techniques: Inhibition, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Analysis, Fluorescence, Reverse Transcription Polymerase Chain Reaction