Western Blot Analysis of Wnt Pathway

Cells were collected at ~90% confluence, lysed in RIPA (20mM Tris pH 7.4, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 100mM NaCl) containing protease and phosphatase inhibitors and analyzed as previously described [11 (link)]. Luciferase were collected in Luciferase assay buffer and resuspended in 2X SDS sample buffer. The following antibodies were used: mouse anti-MAMDC4 1:3 (Wilson Lab), rabbit anti-actin (cat. 4970; CST), mouse anti-total β-catenin (cat. 610153; BD Biosciences), rabbit anti total β-catenin (cat. 8480; CST) rabbit anti-non-phospho (active) β-catenin (cat. 19807; CST), rabbit anti-Axin1 (cat. 2074; CST), rabbit anti-LRP6 (cat. 2560; CST), rabbit anti-phospho-LRP6 (Ser 1490) (cat. 2568; CST), rabbit anti-total GSK (cat. 12456; CST), rabbit anti-phospho-GSK3β (Ser 9) (cat. 5558; CST), rabbit anti-DVL2 (cat. 3224; CST), rabbit anti-MST (cat. 3682; CST), rabbit anti-LATS (cat. 3477; CST), rabbit anti-phospho-LATS (cat. 8654; CST), rabbit anti-YAP (cat. 14074; CST), rabbit anti-phospho YAP (cat. 13008; CST), rabbit anti AMOT (cat. 24550–1; ProteinTech). Immunoblots were imaged using a Licor-Odyssey infrared imager. Protein expression was normalized to actin levels.

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Cox C.M., Wu M.H., Padilla-Rodriguez M., Blum I., Momtaz S., Mitchell S.A, & Wilson J.M. (2024). Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4. PLOS ONE, 19(5), e0296003.

Publication 2024

mouse anti-total β-catenin infrared imager

Corresponding Organization : University of Arizona

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of MAMDC4, actin, total β-catenin, non-phospho (active) β-catenin, Axin1, LRP6, phospho-LRP6 (Ser 1490), total GSK, phospho-GSK3β (Ser 9), DVL2, MST, LATS, phospho-LATS, YAP, phospho-YAP, AMOT

control variables

Cell confluence (~90%)
Lysis buffer (RIPA buffer with protease and phosphatase inhibitors)
Luciferase assay buffer
2X SDS sample buffer

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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