Fig. 3. AMBRA1 affects C-MYC de-phosphorylation at Ser62.
a) Protein extracts of MEFs wild-type (+/+), heterozygous (+/gt) and homozygous (gt/gt) for the gene-trap mutation in the Ambra1 locus were analysed by western blot, using antibodies against c-Myc phosphorylated at the Ser62 (P c-MycS62), total c-Myc and Actin. b) Protein extracts of HeLa cells knocked-down for AMBRA1 (siRNA #1 and #2) or treated with aspecific oligonucleotides (CTRL siRNA), as a control, were analysed by western blot, using antibodies against P C-MYCS62, total C-MYC and ACTIN. c) Immunostaining of c-Myc in +/+ and gt/gt MEFs. Scale bar, 20 μm. Fluorescence intensity per cell was quantified by imagej software, as previously described.53 Data are presented as means±s.e.m. and significance is *P<0.05 (n=100 cells pooled from 3 independent experiments in which 6-8 fields were assessed per experiment). d-e) Cells co-transfected with C-MYC and AMBRA1 or βGal, as a control, were treated with cycloheximide and harvested at the indicated time points. Protein extracts of cells were analysed by western blot, using antibodies against C-MYC, AMBRA1 and ACTIN. Quantification is shown in d. Data are presented as mean±s.d. and significance is *P<0.5 and **P<0.05 (n=3 independent experiments). f) Different phosphatase reactions were performed in vitro, using Pp2a-c extracted from +/+ or gt/gt cells, incubated with the same amount of C-MYC. C-MYC immunoprecipitation plus mouse immunoglobulin mix, instead of anti-Pp2a-c, was used as a negative control. The products of the reactions were analysed by western blot, using antibodies against P C-MYCS62, total C-MYC and Pp2a-c. g) HeLa cells were knocked-down for AMBRA1 (AMBRA1 siRNA) or treated with aspecific oligonucleotides (CTRL siRNA), as a control. Endogenous C-MYC was immunoprecipitated using an anti-C-MYC antibody (IP C-MYC) and mouse immunoglobulins were used as control (IP CTRL). The immunoprecipitated complexes were analysed by western blot with an anti-AMBRA1, anti-C-MYC and anti-PP2A-C antibodies.