- PA3-009 - Invitrogen Antibodies PDIA3 antibody

Submitted references ERp18 regulates activation of ATF6α during unfolded protein response.

Oka OB, van Lith M, Rudolf J, Tungkum W, Pringle MA, Bulleid NJ
The EMBO journal 2019 Aug 1;38(15):e100990

Bromodomain inhibition exerts its therapeutic potential in malignant pleural mesothelioma by promoting immunogenic cell death and changing the tumor immune-environment.

Riganti C, Lingua MF, Salaroglio IC, Falcomatà C, Righi L, Morena D, Picca F, Oddo D, Kopecka J, Pradotto M, Libener R, Orecchia S, Bironzo P, Comunanza V, Bussolino F, Novello S, Scagliotti GV, Di Nicolantonio F, Taulli R
Oncoimmunology 2018;7(3):e1398874

Tumor necrosis factor-α treatment of HepG2 cells mobilizes a cytoplasmic pool of ERp57/1,25D₃-MARRS to the nucleus.

Grindel BJ, Rohe B, Safford SE, Bennett JJ, Farach-Carson MC
Journal of cellular biochemistry 2011 Sep;112(9):2606-15

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of ERp29 in HEK cell lysate using Product # PA3-009.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ERp57 was achieved by transfecting PC3 cells with ERp57 specific siRNAs (Silencer® select Product # s6227 and s6229 ). Western blot analysis (Fig a) was performed using Membrane extract from the ERp57 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-ERp57 Rabbit Polyclonal Antibody (Product # PA3-009, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to ERp57.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of Hep G2 (Lane 1), LNCap (Lane 2), PC-3 (Lane 3), MCF-7(Lane 4), MDA-MB-231 (Lane 5), HeLa (Lane 6), tissue extracts of Mouse testis (Lane 7) and Rat testis (Lane 8). The blot was probed with Anti-ERp57 Rabbit Polyclonal Antibody (Product # PA3-009, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 57 kDa band corresponding to ERp57 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % 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® 2 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)
Main image
Experimental details: Immunofluorescent analysis of ERp57 using anti-ERp57 polyclonal antibody (Product # PA3-009) shows staining in HMVEC Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ERp57 using anti-ERp57 polyclonal antibody (Product # PA3-009) shows staining in NS-1 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ERp57 using anti-ERp57 polyclonal antibody (Product # PA3-009) shows staining in p19 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ERp57 was performed using 70% confluent log phase HepG2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ERp57 Rabbit Polyclonal Antibody (Product # PA3-009) at 1:250 in 0.1% BSA and incubated for overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ERp57 using anti-ERp57 polyclonal antibody (Product # PA3-009) shows staining in HMVEC Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of PDIA3 (ERp57) was performed on frozen sections of e18.5 mouse lung tissue. To expose target proteins, antigen retrieval was performed using Citrate buffer, pH=6 using microwave. Following antigen retrieval, tissues were blocked in 4% Normal Donkey Serum (NDS) for 1-2 hours at room temperature. Tissue samples were then probed with anti-Pdia3/Erp57 antibody (Product # PA3-009) at a dilution of 1:200 (Green) along with anti-Nkx2.1 as epithelial cell marker (Red). Primary antibody incubation was carried out overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBS/0.2% Triton X to remove unbound antibody. Detection was performed using Alexa Fluor conjugated anti-rabbit and anti-mouse IgG secondary antibodies at a dilution of 1:500. Following secondary antibody incubation tissues were washed extensively with PBS/0.2% Triton X and mounted using Prolong Gold anti-fade reagent (Product # P36930). Confocal images were taken on a Nikon A1 LUNA inverted microscope using 60X water or 100X oil objectives. Pdia3 is localized in epithelial cells on co-localization with Nkx2.1. Data courtesy of Dr. Jeff Whitsett's lab.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of PDIA3 (ERp57) was performed on frozen sections of postnatal day 1 mouse lung tissue. To expose target proteins, antigen retrieval was performed using Citrate buffer, pH=6 using microwave. Following antigen retrieval, tissues were blocked in 4% Normal Donkey Serum (NDS) for 1-2 hours at room temperature. Tissue samples were then probed with anti-Pdia3/Erp57 antibody (Product # PA3-009) at a dilution of 1:200 (Green) along with anti-Emcn as endothelial cell marker (Red). Primary antibody incubation was carried out overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBS/0.2% Triton X to remove unbound antibody. Detection was performed using Alexa Fluor conjugated anti-rabbit and anti-mouse IgG secondary antibodies at a dilution of 1:500. Following secondary antibody incubation tissues were washed extensively with PBS/0.2% Triton X and mounted using Prolong Gold anti-fade reagent (Product # P36930). Confocal images were taken on a Nikon A1 LUNA inverted microscope using 60X water or 100X oil objectives. Pdia3 is not expressed in endothelial cells based on non-colocalizaition with endomucin (emcn). Data courtesy of Dr. Jeff Whitsett's lab.