- 710857 - Invitrogen Antibodies TJP3 antibody

Luo M, Zhang L, Yang H, Luo K, Qing C
Molecular medicine reports 2020 Oct;22(4):3429-3439

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Caco-2 (Lane1), MCF-7 (Lane 2) and HT-29 (Lane 3). The blots were probed with ZO-3 Recombinant Rabbit Polyclonal Antibody (Product # 710857, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 140 kDa band corresponding to ZO-3 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence was performed on fixed and permeabilized Caco-2 cells for detection of ZO-3 using Anti-ZO-3 Recombinant Rabbit Polyclonal Antibody (Product # 710857, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of ZO-3 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating junctional localization of ZO-3. Panel e) represents control cells with no primary Antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Interrelation of NEAT1, miR-1321 and TJP3. (A) StarBase website predicted the complementary binding sites between NEAT1 and miR-1321. (B) Transfection efficiency of miR-1321 mimics was detected using RT-qPCR. (C) Targeting relationship of NEAT1 and miR-1321 was identified using dual-luciferase reporter gene assays. (D) StarBase website predicted the complementary binding site between miR-1321 and TJP3. (E) Targeting relationship of miR-1321 and TJP3 was identified using dual-luciferase reporter gene assays. (F) Expression of miR-1321 in ES-2 and A2780 cells was measured using RT-qPCR. (G) Effect of NEAT1 on miR-1321 expression was determined using RT-qPCR. (H) Effect of miR-1321 on expression of TJP3 protein was measured using western blotting. Correlations of (I) lncRNA NEAT1 and miR-1321, (J) miR-1321 and TJP3, and (K) lncRNA NEAT1 and TJP3 on ovarian cancer specimens were investigated using a Pearson Correlation Coefficient. Data are representative of >=3 independent experiments. Data are presented as the mean +- SD. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. TJP3 expression and function in OC. (A) TJP3 protein expression level in normal epithelial cells (IOSE80) and OC cells (SKOV3, OVCAR-3, ES-2 and A2780) were measured using western blotting. (B) Transfection efficiency of si-TJP3 in ES-2 and A2780 cells were detected using western blotting. (C) Proliferative abilities of ES-2 and A2780 cells were measured using Cell Counting Kit-8 assays. (D) E-cadherin, N-cadherin and Vimentin protein expression levels in ES-2 and A2780 cells were assessed using western blotting. (E) Migratory and invasive abilities of ES-2 and A2780 cells were measured using Transwell assays. Scale bar, 100 um. (F) Migratory abilities of ES-2 and A2780 cells were measured using a wound-healing assay. Scale bar, 100 um. All data are representative of >=3 independent experiments. Data are presented as the mean +- SD. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. NEAT1 promotes cell epithelial-mesenchymal transition, migration and invasion of ovarian cancer by regulating TJP3. (A) Transfection efficiency of si-NEAT1 in ES-2 and A2780 cells were detected using reverse transcription-quantitative PCR. (B) Effect of NEAT1 on TJP3 protein expression was measured using western blotting. (C) Proliferative abilities of ES-2 and A2780 cells were measured using Cell Counting Kit-8 assays. (D) E-cadherin, N-cadherin and Vimentin protein expression levels in ES-2 and A2780 cells were assessed using western blotting. (E) Migratory and invasive abilities of ES-2 and A2780 cells were measured using Transwell assays. Scale bar, 100 um. (F) Migratory abilities of ES-2 and A2780 cells were measured using a wound-healing assay. Scale bar, 100 um. All data are representative of >=3 independent experiments. Data are presented as the mean +- SD. *P

710857