Human/Mouse/Rat Axin-1 Antibody

Detection of Axin-1 by Western Blot

Proteasome inhibitor induces PARylation of TNKSs and TNKSi reduces basal PARylation levels. A. SW480 and HEK293 cells were exposed to combinations of TNKSi (G007-LK) and MG132 treatments similar to that shown in legend to Fig 3. The cells were then lysed and extracts analysed by IP assay using a specific antibody against PAR. PARylated forms of TNKS1/2 were then detected by western blot. The IgG control was negative. Pull-down of PARylated proteins revealed an increase in PARylation of TNKS1 by MG132 treatment, and a decrease of PARylation induced by TNKSi. When combined, the MG132 dominated and caused some induction of PARylated TNKS1 in both SW480 and HEK293 cells. Levels of PARylated TNKS2 (and also axin, not shown) were too low for detection. Quantification of band intensities (mean ± SD) was from two separate experiments. B. Total extract western blot shows similar TNKS levels after different treatments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/26930278), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Axin-1 by Immunocytochemistry/ Immunofluorescence

Induction of axin puncta by TNKSi is proteasome-dependent. A. SW480 cells were treated with single or combined doses of tankyrase (5 μM G007LK) and proteasome (20 μM MG132) inhibitors. The MG132 was added for 6 h, either simultaneously with G007-LK for a 6 h treatment, or during the last 6 h of a 24 h G007-LK treatment. As shown in the immunofluorescence images, the addition of MG132 caused a reduction in the % of cells with visible TNKSi-induced axin puncta after 6 h treatment. The 6 h MG132 treatment also caused a modest increase in nuclear staining of axin. The later addition of MG132 (at the end of a 24 h G007-LK treatment) caused the induced axin puncta to relocate to the perinuclear region. B. The number of axin puncta per cell were scored by microscopy and categorized (<5, 5–15 or >15 puncta per cell). As shown, TNKSi induced a high number of puncta per cell (more than 80% of cells scored >15 puncta per cell) and this was blocked at 6 h or reduced after 24 h by MG132 treatment. C. A diagram summarizing the effect of G007-LK +/- MG132 on axin pattern and cellular distribution (shown in green). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/26930278), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Axin-1 by Western Blot

Endocytosis inhibitors cause a decrease in the level of Armadillo/ beta-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R+ cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of beta-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in beta-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3 beta and CK1 alpha were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of beta-catenin. This correlated with a decrease in phosphorylated beta-catenin (pT42/S37/S33 beta-catenin; lane 3), as expected because phosphorylated beta-catenin reflects the activity of the degradation complex (Hernández et al., 2012). By contrast, the (mild) decrease in beta-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated beta-catenin, suggesting that Dyngo-4a impacts on the level of beta-catenin through a mechanism that is independent of the destruction complex. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25236598), licensed under a CC-BY license. Not internally tested by R&D Systems.