Ser45 β catenin

Frozen sections cut at 10 μm thickness were fixed, quenched and blocked as described above for Pax7 immunohistochemistry. Sections were then co‐labelled with mouse anti‐Pax7 (1:500) and rabbit antibodies to active, non‐phosphorylated (Ser45) β‐catenin (1:1500; Cell Signaling Technology, Danvers, MA, USA, cat. no. 19807T; RRID:AB_2650576) overnight in a humidified chamber at 4°C. Sections were subsequently washed and incubated for 30 min with horse anti‐mouse Dylight‐594 (1:200; Vector Laboratories; RRID:AB_2336412) and horse anti‐rabbit Dylight‐488 (1:100; RRID:AB_2336403). Sections were mounted with Prolong Gold antifade mountant containing DNA stain 4′,6‐diamidino‐2‐phenylindole (DAPI; cat. no. P36931; Thermo Fisher Scientific). Cell counts were performed on one, entire, midbelly cross section of each muscle analysed. Cells were first assessed for whether they were Pax7 positive and then assessed for whether they were also active β‐catenin positive. Data are expressed as the percentage of total Pax7⁺ satellite cells that also expressed active β‐catenin (β‐catenin⁺ Pax7⁺/ total Pax7⁺). Cell counts were performed on a Leica TCS‐SP5 confocal microscope.

WB was performed as described [49 (link)]. Antibodies used were the following: CK1α, PARP, Mcl1, Ser45 β-catenin, Ser33/37/Thr41 β-catenin, total β-catenin, Ser473 AKT, total AKT (Cell signaling Technology, MA, USA); Mdm2 (Millipore, Italy), GAPDH (Ambion, USA); α-tubulin (Sigma-Aldrich, Italy); Bak (Merck, MA, USA); Bax, p21, p53 (Becton Dickinson, Italy); Caspase 3 (Enzo Life Science, UK). Images were acquired using the Image Quant LAS 500 chemiluminescence detection system (GE Healthcare, USA). Densitometric analysis was performed with Quantity One software (Bio-Rad, Italy).