Anti tnks1 2

Small intestine organoids were grown in 300μl of Matrigel in one well of a 6-well dish for 3 days post-passage. Organoids were then recovered from the Matrigel using Cell Recovery Solution(ref). Organoid pellets were lysed in 30 μl RIPA buffer. Antibodies used for Western blot were: anti-Apc (Millipore, #5535), anti-Axin1 (CST, #2087), anti-non-phosphorylated β-catenin (CST,), anti-phospho-β-catenin S33/S37/T41 (CST, #9561), total β-catenin (CST, #8480), anti-GSK3 (CST, #9832), anti-actin-HRP (Abcam, #ab49900), anti-Tnks1/2 (Santa Cruz, # sc-365897), anti-GFP (Abcam, #ab13970), Anti-α-Tubulin (Millipore Sigma, # CP06).

The entire coding human TNKS cDNA sequence was fused to the IgG Fc domain by ligating into the pcDNA3 EK FC vector using flanking restriction sites introduced by PCR. Human TNKS-FLAG DNA constructs harboring various mutations were engineered using site-directed mutagenesis. HEK293 or COS1 cells were transfected with the corresponding cDNA construct using Effectene (Qiagen) according to the manufacturers protocol. To chemically inhibit TNKS, cells were treated with the corresponding inhibitor for 24 hours. For immunoblotting, cells were lysed in 1X protein sample loading buffer diluted from Biorad 4X Laemmli protein sample buffer (#1610747), and proteins were separated on SDS-PAGE. For immunoprecipitation or IgG pull-down studies, cleared lysates were mixed with Protein A agarose beads in the presence or absence of 2 μg of desired antibody and rotated for 4 hours at 4°C. Beads were then washed three times with lysis buffer (1% NP40 in PBS). Bound proteins were eluted using 2x protein sample loading buffer and separated on SDS-PAGE. Antibodies were acquired from the following sources: anti-FLAG (Sigma, #A2220), anti-TNKS1/2 (Santa Cruz Biotechnology, #sc-8337), anti-NPT (Millipore, #06-747), and anti-ACTIN (Sigma, #A1978).