Anti actin hrp

Whole pancreas samples were minced and lysed in 600 μl of RIPA buffer by homogenization on the TissueLyser II (Quiagen). Pancreatic organoids were grown in 120 μL of Matrigel in one well of a 12-well dish. Organoids were then recovered from the Matrigel using Cell Recovery Solution. Organoid pellets were lysed in 30 μL RIPA buffer. For MEFs, they were cultured in 6-well plates and lysed in 150 μl of RIPA buffer. Antibodies used for Western blot analysis were: anti-actin-HRP (Abcam #ab49900), anti-α-Tubulin (Millipore Sigma #CP06), anti-pERK 44/42 (Cell Signaling Technology #4370), anti-ERK 44/42 (Cell Signaling Technology #9107), anti-pAKT (ser 473) (Cell Signaling Technology #4060), anti-AKT (Cell Signaling Technology #4691), pMEK ( Cell Signaling Technology #9154), MEK (Cell Signaling Technology #8727), pS6 (Cell Signaling Technology #4858), S6 (Cell Signaling Technology #2317), anti-NF1 (Cell Signaling Technology #14623).

Small intestine organoids were grown in 300ul of Matrigel in 1 well each of a 6-well dish containing 3 mls of growth media for 4 days post-passage, then treated with 125ng/ml Adriamycin for 4 hrs to induce p53 expression. Organoids were then recovered from the Matrigel using several rinses with cold PBS. Organoid pellets were lysed with Lamelli buffer. Antibodies used for Western blot were: anti-APC (1:400, FE9 clone, Millipore #MABC202), anti-non-phosphorylated β-catenin (1:1000, #8814, Cell Signaling Technology), anti-p53 (1:500, #NCL-p53-505, Novocastra) and anti-actin-HRP (1:10,000, #ab49900, Abcam).

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).

Preparation of cellular lysates and subsequent immunoblotting were performed as previously described.^{54 (link)} The antibodies used for the immunoblotting are anti-CD99 (Abcam, # ab75858, EPR3097Y), anti-PARP (Cell Signaling Technology, #9542), anti-Caspase 3 (Cell Signaling Technology (CST), #9662), anti-H2A.X (CST, #7631, D17A3), anti-γH2A.X (CST, #9718, Ser139 - 20E3), anti-ACTIN-HRP (Abcam, #ab49900, AC-15), anti-Rabbit IgG-HRP (Cytiva, # NA934), and anti-Mouse IgG-HRP (Cytiva, # NA9311). Enhanced chemiluminescence (ECL) signal on PVDF membranes was captured by using LI-COR Odyssey XF imaging system (LI-COR Biosciences) and densitometry analysis of the protein bands were performed by using Image Studio software (LI-COR Biosciences).