- PA5-29884 - Invitrogen Antibodies UHRF1 antibody

Submitted references The microRNA signature of patients with sunitinib failure: regulation of UHRF1 pathways by microRNA-101 in renal cell carcinoma.

Goto Y, Kurozumi A, Nohata N, Kojima S, Matsushita R, Yoshino H, Yamazaki K, Ishida Y, Ichikawa T, Naya Y, Seki N
Oncotarget 2016 Sep 13;7(37):59070-59086

Regulation of UHRF1 by dual-strand tumor-suppressor microRNA-145 (miR-145-5p and miR-145-3p): Inhibition of bladder cancer cell aggressiveness.

Matsushita R, Yoshino H, Enokida H, Goto Y, Miyamoto K, Yonemori M, Inoguchi S, Nakagawa M, Seki N
Oncotarget 2016 May 10;7(19):28460-87

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of UHRF1 using 30 µg of HCT116 lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a UHRF1 polyclonal antibody (Product # PA5-29884) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using UHRF1 Polyclonal Antibody (Product # PA5-29884). Mouse tissue extract (50 µg) was separated by 7.5% SDS-PAGE, and the membrane was blotted with UHRF1 Polyclonal Antibody (Product # PA5-29884) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of UHRF1 was achieved by transfecting MCF7 with UHRF1 specific siRNAs (Silencer® select Product # s70719, s70720). Western blot analysis (Fig. a) was performed using whole cell extracts from the UHRF1 knockdown cells (lane 2) and non-targeting scrambled siRNA transfected cells (lane 1). The blot was probed with UHRF1 Polyclonal Antibody (Product # PA5-29884, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 UHRF1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-UHRF1 Polyclonal Antibody(Product # PA5-29884) and a 90 kDa band corresponding to UHRF1 was observed along with uncharacterized bands(*) across cell lines . Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), HeLa (Lane 2) and MDA-MB-231 (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of UHRF1 in paraformaldehyde-fixed HeLa cells using a UHRF1 polyclonal antibody (Product # PA5-29884) (Green) at a 1:500 dilution. Alpha-tubulin filaments were labeled with Product # PA5-29281 (Red) at a 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of UHRF1 was performed in A549 cells fixed in 4% paraformaldehyde at RT for 15 min. Green: UHRF1 Polyclonal Antibody (Product # PA5-29884) diluted at 1:250. Red: alpha Tubulin, a cytoskeleton marker.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of UHRF1 was performed using 70 confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with UHRF1 Polyclonal Antibody (Product # PA5-29884) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing Predominant Nuclear along with faint cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: UHRF1 Polyclonal Antibody detects UHRF1 protein at on HBL435 xenograft by immunohistochemical analysis. Sample: Paraffin-embedded HBL435 xenograft. UHRF1 Polyclonal Antibody (Product # PA5-29884) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Cross-linked ChIP was performed with HCT116 chromatin extract and 5 µg of either control rabbit IgG or UHRF1 Polyclonal Antibody (Product # PA5-29884). The precipitated DNA was detected by PCR with primer set targeting to PPARG promoter.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous UHRF1 protein using UHRF1 Antibody: ChIP was performed using UHRF1 Polyclonal Antibody (Product # PA5-29884, 5 µg) on sheared chromatin from HCT 116 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to MDR1-TSS, UCHL1, PPARG, ADAM19, SFRP1 (active) and MDR1-Exon6 and SAT alpha satellite repeats (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of UHRF1 protein from HeLa whole cell extract using 5 µg of UHRF1 Polyclonal Antibody (Product # PA5-29884). Western blot analysis was performed using UHRF1 Polyclonal Antibody (Product # PA5-29884).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: UHRF1 Polyclonal Antibody immunoprecipitates UHRF1 protein in IP experiments. IP Sample: HeLa whole cell lysate/extract A. 40 µg HeLa whole cell lysate/extract B. Control with 2 µg of preimmune rabbit IgG C. Immunoprecipitation of UHRF1 protein by 2 µg of UHRF1 Polyclonal Antibody (Product # PA5-29884) 5% SDS-PAGE The immunoprecipitated UHRF1 protein was detected by UHRF1 Polyclonal Antibody (Product # PA5-29884) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 Immunohistochemical staining of UHRF1 in BC clinical specimens UHRF1 was expressed more strongly in several cancer lesions than in noncancerous tissues. Left panel, original magnification x40; Right panel, original magnification x200. ( A ) Positively stained tumor lesion (High grade, T2bN0M0), ( B ) Positively stained tumor lesion (High grade, T1N0M0), ( C ) Positively stained tumor lesion (Low grade, T3N0M0), ( D ) Negative staining in normal bladder tissue.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Direct regulation of UHRF1 by miR-145-5p and miR-145-3p ( A ) UHRF1 mRNA expression was evaluated by qRT-PCR in T24 and BOY 72 hours after transfection with miR-145-5p and miR-145-3p . GUSB was used as an internal control. * P = 0.0036 and ** P < 0.0001. ( B ) UHRF1 protein expression was evaluated by Western blot analyses in T24 and BOY 72-96 hours after transfection with miR-145-5p or miR-145-3p . GAPDH was used as a loading control. ( C ) miR-145-5p and miR-145-3p binding sites in the 3' UTR of UHRF1 mRNA. Dual Luciferase reporter assays using vectors encoding putative miR-145-5p and miR-145-3p target sites of the UHRF 3' UTR (positions 1,179-1,198 and 287-292, respectively) for both wild-type and deleted regions. Normalized data were calculated as ratios of Renilla /firefly luciferase activities. * P < 0.0001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 UHRF1 mRNA and protein expression after si-UHRF1 transfection and effects of UHRF1 silencing in BC cell lines ( A ) UHRF1 mRNA expression was evaluated by qRT-PCR in T24 and BOY 72 hours after transfection with si-UHRF1 -1 and si-UHRF1 -2. GUSB was used as an internal control. ( B ) UHRF1 protein expression was evaluated by Western blot analysis in T24 and BOY 72 - 96 hours after transfection with miR-145-5p or miR-145-3p . GAPDH was used as a loading control. ( C ) Cell proliferation was determined with the XTT assays 72 hours after transfection with 10 nM si-UHRF1 -1 or si-UHRF1 -2. * P < 0.0001. ( D ) Cell migration activity was determined by wound-healing assays. * P < 0.0001. ( E ) Cell invasion activity was determined using Matrigel invasion assays. * P < 0.0001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 miR-101 directly downregulated UHRF1 expression in RCC cells ( A ) UHRF1 mRNA expression 72 h after transfection with miR-101 . GUSB was used as an internal control. ( B ) UHRF1 protein expression 72 h after transfection with miR-101 . GAPDH was used as a loading control. ( C ) miR-101 binding sites in UHRF1 mRNA. Luciferase reporter assays were carried out using a vector encoding the putative miR-101 target site in the UHRF1 3'-UTR (position 1030-1036) for wild-type and deletion constructs. * P < 0.002, ** P < 0.0001. The bars indicate SDs.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Effects of UHRF1 knockdown in RCC cells and impact of UHRF1 expression on clinical RCC specimens ( A ) UHRF1 mRNA expression was determined at 72 h after transfection with si-UHRF1 . GUSB was used as an internal control. ( B ) UHRF1 protein expression was evaluated by western blotting at 72 h after transfection with si-UHRF1 . GAPDH was used as a loading control. * P < 0.0001. The bars indicate SDs. ( C ) Cell proliferation was assessed 72 h after transfection with si-UHRF1 using XTT assays. ( D ) Cell migration was assessed 48 h after transfection with si-UHRF1 using uncoated Transwell polycarbonate membrane filters. ( E ) Cell invasion was assessed 48 h after transfection with si-UHRF1 using Matrigel invasion assays. * P < 0.0001. The bars indicate SDs.

PA5-29884

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