Mouse α ubiquitin fk 2

Manufactured by R&D Systems

Mouse α-ubiquitin (FK-2) is a monoclonal antibody that recognizes the polyubiquitin conjugates in mammalian cells. It is a useful tool for the detection of ubiquitinated proteins.

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Ubiquitin (146 citations)

2 protocols using mouse α ubiquitin fk 2

Immunostaining and Protein Analysis of RhoGTPases

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The following antibodies were used for immunostaining: α-VE-cadherin XP (#2500; Cell Signaling Technologies, Danvers, MA) and a-RhoB (#sc-8048; Santa Cruz Biotechnology, Santa Cruz, CA). Alexa Fluor 488 secondary antibody (anti-rabbit and anti-mouse) and Alexa Fluor 647 secondary antibody (anti-rabbit) (all Invitrogen) were the secondary antibodies.
The following antibodies were used for protein analysis: ß-actin (Merck), LDLR (Biovision), and SQLE (Proteintech). α-p44/42 MAPK (ERK1/2) (#9102), α-RhoA (#2117), α-RhoC (#3430), and α-GAPDH (#2118; Cell Signaling Technologies), α-RhoB (#sc-180, #sc-8048; Santa Cruz Biotechnology), α-HA (H3663), and α-vinculin (V4139; Sigma-Aldrich), α-Fbxw7 (ab171961; Abcam) and mouse α-ubiquitin (FK-2; Boston Biochem). HRP-conjugated goat–anti-rabbit antibody and goat–anti-mouse (Dako) were used as secondary antibodies.
For inhibition of geranylgeranylation and farnesylation, GGTI-298 and FTI-277 HCL (both Selleck Chemicals) were used. For normalization of prenylation in the FBXW7 knockdown cells, GGPP or FPP were applied (Sigma).
The siRNAs used were ON-TARGET plus Nontargeting pool (siNT), ON-TARGET plus Human FBXW7 pool (siFBXW7), ON-TARGET plus Human RHOA siRNA pool (siRhoA), ON-TARGET plus Human RHOB siRNA pool (siRhoB), and ON-TARGET plus Human FBXW7 siRNA—Set of 4 Upgrade (all Dharmacon/GE-Healthcare, Lafayette, CO).

Pronk M.C., Majolée J., Loregger A., van Bezu J.S., Zelcer N., Hordijk P.L, & Kovačević I. (2019). FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB. Molecular Biology of the Cell, 30(5), 607-621.

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Antibody Panel for Cell Signaling

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The following primary antibodies were used in this study: mouse αVE–cadherin/CD144–Alexa Fluor 647 (BD), rabbit αErk1/2 (Santa Cruz), mouse αpErk1/2 (Santa Cruz), rabbit αpMyosin LC2 Thr18/Ser19 (Cell Signaling), rabbit αpPAK1/2/3 Ser141 (Invitrogen), rabbit αvinculin (Sigma), rabbit αGAPDH (Cell Signaling), mouse αHA (Sigma), rabbit αLAMP1 (Cell Signaling), rabbit αKCTD10 (Sigma), rabbit αPaxillin Tyr31 (Sigma), mouse αRac1 (BD), rabbit αRhoC (Cell Signaling), rabbit αRhoA (Cell Signaling), rabbit αRhoB (Santa Cruz), mouse αRhoB (Santa Cruz), mouse αUbiquitin (FK-2; Boston Biochem), rabbit αUbiquitin, Lys63-specific (Apu3; Millipore), rabbit αCullin-3 (Cell Signaling), mouse αCullin-2 (BD), mouse αCullin-1 (Santa Cruz), rabbit αKCTD10 (Sigma), rat αmouseCD29 (9eg7; BD), rabbit αIntegrin α-5 (Abcam), and rabbit p-NF-κB p65 (Ser536; Cell Signaling). Mouse anti–HA agarose was purchased from Sigma.

Kovačević I., Sakaue T., Majoleé J., Pronk M.C., Maekawa M., Geerts D., Fernandez-Borja M., Higashiyama S, & Hordijk P.L. (2018). The Cullin-3–Rbx1–KCTD10 complex controls endothelial barrier function via K63 ubiquitination of RhoB. The Journal of Cell Biology, 217(3), 1015-1032.

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