Anti ha mouse primary antibody

For protein extraction and immunoblots, mouse tissues or organoids were lysed on ice in RIPA lysis buffer (50mM Tris HCl pH 7,4, 150 mM NaCl, 1% NP40, 0,25% sodium deoxycholate) containing complete protease inhibitors (Roche). For organoids beforehand, lysis Matrigel was removed using cell recovery solution (Corning) as described in manufacturer’s protocol. Samples were then boiled for 10 min and cleared by centrifugation. Equal amounts of lysate protein were separated performing SDS-PAGE. Western blotting was performed with anti HA mouse primary antibody (Roche) and anti actin rabbit primary antibody (SIGMA) and then with secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase (Sigma and Amersham Biosciences, respectively). Visualization was achieved with the Western Lightning Plus-ECL solution (PerkinElmer).

Fifty micrograms of a plasmid containing the HA-PRL-3 human cDNA under the control of the doxycycline (dox) responsive promoter (tetO) (see plasmids) was inserted downstream of the Col1A1 locus (Fig s1A) by co-electroporation with 25 μg of a Flpe recombinase containing vector into KH2-ES cells [35 (link)]. After ES cell electroporation, three positive clones were able to grow in presence of 150 μg/mL hygromycin. These clones (A1, B1, and B2) were expanded, and HA-PRL-3 protein expression was checked by western blot after dox treatment with anti HA mouse primary antibody (Roche) (Fig s1B and Sup1). Subsequently, clones A1 and B1 were independently microinjected into blastocysts fertilized C57BL/6J embryos. Thus, two bi-transgenic (HA-PRL-3 het/R26-rtTA het) mice from these two independent litters were used to expand the two TG mouse colonies separately, corresponding to founders A1 and B1. In order to discard possible random transgenesis, the phenotype of each mouse colony was independently analyzed, and the same phenotype was observed in both.