Protein lysates were run on Tris-HCl, 1mm Criterion Precast gels (Bio-Rad) or NuPAGE Bis-Tris gels (Novex) gels at a constant voltage. Proteins were transferred onto Immobilon-P transfer membranes (Millipore) at a constant amperage. Before staining with primary antibodies, blots were blocked in 5% non-fat dry milk (Santa Cruz) or 5% BSA in TBS-T 0.1% for 30 minutes.
For protein detection primary antibodies detecting CK1α (C-19, Santa Cruz or Abcam ab108296), β-catenin (Cell Signaling #9587 and #8480), HA (HRP-conjugate, Miltenyi, GG8-1F3.3), FLAG (M2, HRP-conjugate Sigma Aldrich), ubiquitin conjugates (FK2, HRP-conjugate Enzo Life Sciences), Actin (HRP-conjugate, Abcam), β-tubulin (Cell Signaling #2146) and GAPDH (Santa Cruz sc-47724) were used. Secondary antibodies were HRP conjugated Bovine anti-Goat (Jackson ImmunoResearch) and HRP conjugated donkey anti-rabbit (GE Healthcare). SuperSignal (Thermo Scientific) chemi-luminescent substrate was used for detection. For re-probing, blots were stripped in Restore Western Blot Stripping Buffer (Thermo Scientific), activated in methanol, and re-blocked.
For protein detection primary antibodies detecting CK1α (C-19, Santa Cruz or Abcam ab108296), β-catenin (Cell Signaling #9587 and #8480), HA (HRP-conjugate, Miltenyi, GG8-1F3.3), FLAG (M2, HRP-conjugate Sigma Aldrich), ubiquitin conjugates (FK2, HRP-conjugate Enzo Life Sciences), Actin (HRP-conjugate, Abcam), β-tubulin (Cell Signaling #2146) and GAPDH (Santa Cruz sc-47724) were used. Secondary antibodies were HRP conjugated Bovine anti-Goat (Jackson ImmunoResearch) and HRP conjugated donkey anti-rabbit (GE Healthcare). SuperSignal (Thermo Scientific) chemi-luminescent substrate was used for detection. For re-probing, blots were stripped in Restore Western Blot Stripping Buffer (Thermo Scientific), activated in methanol, and re-blocked.