- PA5-99680 - Invitrogen Antibodies OTUB2 antibody

Ouyang S, Zeng Z, Liu Z, Zhang Z, Sun J, Wang X, Ma M, Ye X, Yu J, Kang W
Cell death discovery 2022 Feb 2;8(1):45

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of OTUB2 in HepG2 cell lysate. Samples were incubated with OTUB2 polyclonal antibody (Product # PA5-99680).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of OTUB2 in various samples (Lane 1: 293 treated with blocking peptide, Lane 2: 293, Lane 3: HeLa). Samples were incubated with OTUB2 polyclonal antibody (Product # PA5-99680).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of OTUB2 in COLO205 cells. Samples were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% serum (45 min at 25°C) incubated with OTUB2 polyclonal antibody (Product # PA5-99680) using a dilution of 1:200 (1 hr, 37°C), and followed by goat anti-rabbit IgG Alexa Fluor 594 at a dilution of 1:600.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of paraffin-embedded OTUB2 in human liver tissue. Antigen retrieval was performed using citrate buffer. Samples were blocked with blocking buffer (1.5 hr, 22°C), incubated with OTUB2 polyclonal antibody (Product # PA5-99680) using a dilution of 1:100 (1.5 hr, 22°C), followed by HRP conjugated goat anti-rabbit.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1 OTUB2 and KRT80 are expressed in gastric cancer tissues. a Real-time PCR analysis of the expression of OTUB2 and KRT80 in 32 primary gastric cancer tissues and paired normal tissues. The levels of mRNA expression were normalized to those of beta-actin. b Western blotting was used to determine the expression levels of OTUB2 and KRT80 proteins in eight representative gastric cancer specimens; ""N"" denotes adjacent normal tissue and ""T"" denotes tumor tissue. At least three separate experiments are represented by the data.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 OTUB2-promoting gastric cancer cell proliferation is coincident with KRT80 in vitro. a Western blotting analysis of OTUB2 and KRT80 expression in four gastric cancer cell lines. b - f Western blot analysis, CCK-8 assay, plate colony formation assay, and quantification of OTUB2 and KRT80 expression and cellular growth in AGS cells and MKN45 cells with stable OTUB2-knockdown, KRT80-knockdown, KRT80-overexpression, KRT80-overexpression, and rescued-KRT80 respectively. The number of gastric cancer cell clones was quantified. Error bars represent the mean +- SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns not significant (Student's t -test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 OTUB2 interacts with and stabilizes KRT80. a Reciprocal co-IP of OTUB2 and KRT80. Plasmids encoding Myc-tagged OTUB2 and Flag-tagged KRT80 were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag or anti-Myc antibodies, followed by western blotting as indicated. b Endogenous co-IP of OTUB2 and KRT80 in AGS cells. Anti-OTUB2 and anti-KRT80 antibodies were used to immunoprecipitate cell lysates, which were then identified by western blotting. c Flag-tagged KRT80 (0.4 mug) was co-transfected into HEK293T cells with the specified doses of Myc-OTUB2 (0.25, 0.5, 0.75, 1, and 1.5 mug) and Myc vector. After 48 h, the transfected cells were harvested and western blotting analysis was performed. d Effects of OTUB2 on the degradation of KRT80 in HEK293T cells. Flag-tagged KRT80 was co-transfected into HEK293T cells with or without Myc-tagged OTUB2 or OTUB2-C51S and treated with CHX at various time points (0, 4, 8, and 12 h) prior to harvesting. The amount of Flag-tagged KRT80 protein was determined using an anti-Flag antibody. e Effects of OTUB2 on the degradation of KRT80 in gastric cancer cells. AGS-shck/shOTUB2 cells were exposed to CHX (20 mug/mL) and harvested at the indicated times. KRT80 was measured using western blotting. KRT80 was stabilized when cells were co-treated with CHX and MG132 (20 muM).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 OTUB2 regulates KRT80, thereby activating the Akt signaling pathway, which results in the tumorigenesis and proliferation of GC in vivo. a Reduced Akt phosphorylation levels were detected by western blotting in OTUB2-knockdown AGS cells. Total Akt, mTOR, p70 S6K, and GAPDH served as controls. Increased Akt phosphorylation levels, as detected by western blotting, in KRT80-overexpressing MKN45 cells. b - e Effect of OTUB2-knockdown, KRT80 rescued and control AGS cells ( b ) or KRT80-overexpression MKN45 cells ( d ) on GC tumorigenesis in vivo. The volume of subcutaneous tumors was measured ( n = 5 or n = 3). * P < 0.05; ** P < 0.01 (Student's t -test).

PA5-99680