evaluation software aida image analyser 2d densitometry program  (Raytest GmbH)

Journal: The Febs Journal

Article Title: AXIN2 promotes degradation of AXIN1 through tankyrase in colorectal cancer cells

doi: 10.1111/febs.17226

Figure Lengend Snippet: The increase in AXIN1 overcompensates for the decrease of AXIN2. (A) Western blotting for endogenous AXIN1 and AXIN2 in lysates of SW480 cells that were untreated (−) or treated with 100 n m G007‐LK overnight, and for GFP‐AXIN2 and GFP‐AXIN1 in lysates of transfected SW480 cells, which were loaded as 1 : 10 diluted, 1 : 2 diluted and undiluted, using the antibodies indicated on the right. Numbers below the blots indicate the intensities of the respective protein bands determined by 2D densitometry, and were used for the exemplary calculation in (B). (B) Calculation of the ratio of the protein level of AXIN1 relative to the protein level of AXIN2 in SW480 cells, based on the numbers determined in (A). (C) Quantification of the AXIN1/AXIN2 protein ratio in SW480 cells from six independent experiments, as shown in (A, B), as the mean ± SEM. (D) Pie charts illustrating the relative levels of AXIN1 (blue) and AXIN2 (green) in parental SW480 WT cells (SW480), SW480 AXIN2 knockout (AXIN2 −/−) and knockdown cells (siAXIN2). The sizes of the pie charts represent the combined pool of AXIN1 and AXIN2 (Total). The SW480 WT chart reflects the mean AXIN1/AXIN2 ratio of 3.6 as determined in (C), and the combined pool of AXIN1 and AXIN2 was set to 100%. The amounts of AXIN1 and AXIN2 in the AXIN2 knockout and knockdown cells were calculated trough the fold changes relative to the WT cells (provided in square brackets). These fold changes of AXIN1 in the knockout cells (mean fold change of both clones) and of AXIN1 and AXIN2 in the knockdown cells were calculated based on the data shown in Fig. (for details, see Table ).

Article Snippet: Band intensities were quantified with AIDA 2D densitometry (Elysia‐Raytest, Angleur, Belgium).

Techniques: Western Blot, Transfection, Knock-Out, Knockdown, Clone Assay

Journal: The Febs Journal

Article Title: AXIN2 promotes degradation of AXIN1 through tankyrase in colorectal cancer cells

doi: 10.1111/febs.17226

Figure Lengend Snippet: Loss of AXIN2 increases Wnt signaling and AXIN1 levels. (A–D) Analysis of parental SW480 WT cells (+/+ #1, black bars), one WT control clone (+/+ #2, black bars) and two AXIN2 knockout clones (AXIN2 −/− #1 and #2, green bars). (E–I) Analysis of SW480 cells transfected with control siRNA (−, black bars) or siAXIN2 (AXIN2 knockdown cells, green bars). (A, E) Relative luciferase activity reporting β‐catenin‐dependent transcription in the indicated cells ( n = 4). (B, F) Western blotting for endogenous AXIN1, AXIN2, β‐catenin and α‐tubulin (loading control) in hypotonic lysates of the indicated cells. (C, D, G–I) Relative protein levels of β‐catenin (C, G), AXIN1 (D, H) or AXIN2 (I) normalized to α‐tubulin (loading control), as determined by 2D densitometry analysis of protein bands from four independent experiments [as shown in (B)] (C, D) and [as shown in (F)] (G–I; n = 4). Results are the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t ‐test).

Article Snippet: Band intensities were quantified with AIDA 2D densitometry (Elysia‐Raytest, Angleur, Belgium).

Techniques: Control, Knock-Out, Clone Assay, Transfection, Knockdown, Luciferase, Activity Assay, Western Blot

Journal: The Febs Journal

Article Title: AXIN2 promotes degradation of AXIN1 through tankyrase in colorectal cancer cells

doi: 10.1111/febs.17226

Figure Lengend Snippet: AXIN2 loss disrupts TNKS‐mediated regulation of Wnt signaling and AXIN1 levels. (A) Relative luciferase activity reporting β‐catenin‐dependent transcription in parental SW480 cells (+/+ #1, black bars), one WT control clone (+/+ #2, black bars) and two AXIN2 knockout clones (AXIN2 −/− #1 and #2, green bars), which were untreated (0) or treated with 10 or 100 n m of the TNKS small‐molecule inhibitor G007‐LK overnight, as indicated ( n = 4). The activity in untreated cells (0) was set to 100% for every clone to facilitate comparisons of the G007‐LK effect. (B) Western blotting for endogenous β‐catenin, AXIN1, AXIN2 and α‐tubulin (loading control) in hypotonic lysates of the indicated cells without or with 100 n m G007‐LK treatment overnight (+). (C–E) Relative protein levels of β‐catenin (C), AXIN1 (D) or AXIN2 (E) normalized to α‐tubulin (loading control), as determined by 2D densitometry analysis of protein bands from four independent experiments as in (B) ( n = 4). The β‐catenin levels (C) in untreated cells were set to 100% for every clone to facilitate comparisons of the G007‐LK effect. (A, C–E) Results are shown as the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001; ns (not significant), P > 0.05 (Student's t ‐test). (F) Pie charts illustrating the relative levels of AXIN1 (blue) and AXIN2 (green) in the indicated cells without (untreated) or with 100 n m G007‐LK treatment overnight. The sizes of the pie charts represent the combined pool of AXIN1 and AXIN2 (Total). The parental SW480 WT chart (+/+ #1) reflects the mean AXIN1/AXIN2 ratio as determined in Fig. , and the combined pool of AXIN1 and AXIN2 was set to 100%. The amounts of AXIN1 and AXIN2 in the other pie charts were calculated through the fold changes relative to the WT cells (provided in square brackets), as determined in (D) and (E) (for details of the calculation, see Table ).

Article Snippet: Band intensities were quantified with AIDA 2D densitometry (Elysia‐Raytest, Angleur, Belgium).

Techniques: Luciferase, Activity Assay, Control, Knock-Out, Clone Assay, Western Blot

Journal: The Febs Journal

Article Title: AXIN2 promotes degradation of AXIN1 through tankyrase in colorectal cancer cells

doi: 10.1111/febs.17226

Figure Lengend Snippet: TNKS inhibitors depend on AXIN2 for regulating β‐catenin and AXIN1 levels. (A, E, I, M) Western blotting for endogenous β‐catenin, AXIN1, AXIN2 and α‐tubulin (loading control) in lysates of SW480 WT cells (+/+) and AXIN2 knockout cells (−/−), which were untreated or treated with TNKS small‐molecule inhibitors (IWR‐1, MSC2504877, OM‐153, XAV939) overnight, as indicated. Dotted vertical lines in (M) indicate splicing of the blots. (B–D, F–H, J–L, N–P) Relative protein levels of β‐catenin (B, F, J, N), AXIN1 (C, G, K, O) or AXIN2 (D, H, L, P) normalized to α‐tubulin (loading control), as determined by 2D densitometry analysis of protein bands from four independent experiments as in (A), (E), (I) and (M), respectively ( n = 4). The β‐catenin levels (B, F, J, N) in untreated cells were set to 100% for WT and knockout cells to facilitate comparisons of the TNKS inhibitor effect. Results are shown as the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001; ns (not significant), P > 0.05 (Student's t ‐test).

Article Snippet: Band intensities were quantified with AIDA 2D densitometry (Elysia‐Raytest, Angleur, Belgium).

Techniques: Western Blot, Control, Knock-Out

Journal: The Febs Journal

Article Title: AXIN2 promotes degradation of AXIN1 through tankyrase in colorectal cancer cells

doi: 10.1111/febs.17226

Figure Lengend Snippet: AXIN2 promotes degradation of AXIN1. (A) Western blotting for endogenous AXIN1 and α‐tubulin (loading control) in lysates of parental SW480 WT cells (+/+) and AXIN2 knockout cells (−/−), which were untreated (0) or treated with 100 μ m CHX for 4 or 8 h. (B) Relative protein levels of AXIN1 normalized to α‐tubulin (loading control), as determined by 2D densitometry analysis of protein bands from four independent experiments as in (A). (C) Schematic to scale representation of rat AXIN1 WT (1–827) with its binding sites for APC (RGS), GSK3B (GSK), β‐catenin (β) and the polymerizing DIX domain. Amino acids 1–89 are magnified below, and the two TNKS binding segments and four phosphorylation sites that interfere with TNKS‐mediated degradation of AXIN1 are highlighted in red and orange, respectively. (D, F) Western blotting for GFP‐AXIN1 WT and 4xD, Flag‐TNKS and Flag‐AXIN2 in lysates of U2OS cells (D), or for GFP‐AXIN1 89‐827, GFP‐TNKS and Flag‐AXIN2 in lysates of U2OS and HEK293T cells (F), as indicated. Cells have been transiently transfected as indicated above the blots. α‐Tubulin: loading control. Asterisk: unspecific band. (E, G) Relative protein levels of AXIN1 WT and 4xD (E) and of AXIN1 WT and 89–827 (G) normalized to α‐tubulin (loading control) in cells co‐expressing TNKS without or with additional co‐expression of AXIN2, as determined by 2D densitometry analysis of protein bands from three independent experiments ( n = 3) [as shown in (D)] (E) and [as shown in (F)] (G). (B, E, G) Results are shown as the mean ± SEM, * P < 0.05, ** P < 0.01 (Student's t ‐test).

Article Snippet: Band intensities were quantified with AIDA 2D densitometry (Elysia‐Raytest, Angleur, Belgium).

Techniques: Western Blot, Control, Knock-Out, Binding Assay, Phospho-proteomics, Transfection, Expressing