- 37-2100 - Invitrogen Antibodies PEBP1 antibody

Submitted references RKIP Regulates Differentiation-Related Features in Melanocytic Cells.

Penas C, Apraiz A, Muñoa I, Arroyo-Berdugo Y, Rasero J, Ezkurra PA, Velasco V, Subiran N, Bosserhoff AK, Alonso S, Asumendi A, Boyano MD
Cancers 2020 Jun 3;12(6)

Proteomic characterization of differentially expressed proteins in breast cancer: Expression of hnRNP H1, RKIP and GRP78 is strongly associated with HER-2/neu status.

Zhang D, Tai LK, Wong LL, Putti TC, Sethi SK, Teh M, Koay ES
Proteomics. Clinical applications 2008 Jan;2(1):99-107

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of mouse brain homogenates using Ms anti-RKIP (C-term) (Product # 37-2100).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of PEBP1 was achieved by transfecting HeLa cells with PEBP1 specific siRNAs (Silencer® select Product # s9982, s9983). Western blot analysis (Fig a) was performed using membrane enriched extracts from the PEBP1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-PEBP1 Monoclonal Antibody (Product # 37-2100, 2 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PEBP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane-enriched extracts (30 µg lysate) of HEK-293 (Lane 1), HCT 116 (Lane 2), C2C12 (Lane 3), 3T3-L1 (Lane 4), COS-7 (Lane 5), HeLa (Lane 6), A-431 (Lane 7), and tissue extract (30 µg lysate) of mouse brain (Lane 8). The blots were probed with Mouse Anti-RKIP (C-term) Monoclonal Antibody (Product # 37-2100, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 21 kDa band corresponding to PEBP1 (RKIP C-term) was observed across the cell lines and tissues tested. The 42 kDa band observed in HEK-293 (Lane 1) and mouse brain (Lane 7) could potentially be PEBP1 dimers, caused by the PKC-mediated phosphorylation of PEBP1, as reported in literature. 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® 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: Immunofluorescence analysis of PEBP1 was performed using 70% confluent log phase HCT-116 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 Prostate Specific Acid Phosphatase (PASE/4LJ) Mouse Monoclonal Antibody (3721) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the control without primary antibody. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 RKIP expression in cell culture of primary melanocytes and melanomas. ( a ) RKIP mRNA expression in primary melanocytes and melanoma cell lines. RNA from three human melanocytes (dark green) lightly (HEMn-LP), moderately (HEMn-MP), and darkly (HEMn-DP) pigmented neonatal foreskin lines together with four primary melanomas cell lines (light green) and seven metastatic melanomas cell lines (light blue) were analyzed by RT-qPCR. ( b ) RKIP protein expression in the three melanocytes cell lines and five melanoma cell lines assessed by Western Blot. Tubulin expression was used as loading control. Figure is representative of three independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Modulation of RKIP expression in primary melanoma cell lines. ( a ) RKIP mRNA levels in RKIP-downregulated A375 and MelHO primary melanoma cell lines. A375 and MelHO cells were transduced with RKIP shRNA Lentiviral Particles or Control shRNA Lentiviral Particles following the manufacturer's instruction. Two days after infection, the cells were selected with Puromycin to get stable cell lines; ( b ) Western Blot assay showed the RKIP-downregulation in A375 and MelHO melanoma cells; ( c ) Proliferation rate in A375 and MelHO primary melanomas after RKIP downregulation. The viability of control melanoma cells (with an empty vector) and stable RKIP transfected clones were subjected to XTT assays for 24, 48, and 72 h. Results of each experiment are expressed related to the values obtained for the transfection control. Data is given as a mean +- SD of at least three experiments of different transfection; ( d ) Fold change on wound healing rate in A375 and MelHO primary melanoma after RKIP downregulation; ( e ) Fold change on active migration rate in presence of collagen in primary melanoma after RKIP downregulation. The histograms in ( d ) and ( e ) show the average of three independent assays with six replicates per assay and representative pictures have been included. * p -value < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Modulation of RKIP expression in MeWO and A2058 metastatic melanoma cell lines. ( a ) RKIP mRNA levels in RKIP-upregulated MeWO and A2058 metastatic melanoma cell lines. A2058 and MeWO cell lines were transfected with overexpressing plasmid for RKIP (pRKIP) or empty plasmid (pCTR) using Lipofectamine 2000 as transfection agent. All of the transfection experiments were performed with 500 ng of DNA. ( b ) Western Blot assay showed the RKIP-upregulation in MeWO and A2058 melanoma cells; ( c ) Fold change on wound healing rate in MeWO and A2058 metastatic melanoma after RKIP upregulation. The experimental assays were performed at least after 24 h of RKIP transfection; ( d ) Fold change on active migration rate in presence of collagen in metastatic melanoma after RKIP upregulation. The histograms in ( c ) and ( d ) show the average of three independent assays with six replicates per assay and representative pictures have been included. * p -value < 0.05.

37-2100

Antibody data