Western lightning chemiluminescence ecl reagents

The human p53 isoform constructs used in this report were previously described (Bourdon et al., 2005 (link)). They were sub-cloned into the EcoRI and BamHI sites of pEGFPC1 (Clonetech) to obtain GFP-tagged proteins or in the BamHI and EcoRI sites of pLPCmyc to obtain myc-tagged proteins. Constructs were amplified using the Nucleobond PC 500 kit (Macherey-Nagel) according to the manufacturer’s instructions. Human Δ133p53 isoforms (α, β and γ) were cloned into pMSCVhyg (Clontech Laboratories) plasmid for retrovirus production. pSIREN-Luc (luciferase)-shRNA was purchased from Clontech. The mouse anti-E-Cadherin (clone 36) and the mouse anti-Beta1-Integrin, were purchased from BD-transduction laboratories and were diluted at 1/400° and 1/250°, respectively. The mouse N-Cadherin (clone 32/BD Transduction laboratories), and the rabbit polyclonal anti-GFP (Invitrogen, Life Technologies, A-6455) were used at 1/400°, 1/250°, 1/300° and 1/2000°, respectively.
The sheep pantropic p53 antibody Sapu was diluted at 1/5000°. The rabbit polyclonal KJC8 antibody specific of the β p53 isoforms was diluted at 1/4000° (Bourdon et al., 2005 (link)). The Horse-Radish Peroxidase (HRP)-conjugated anti-IgG antibodies were purchased from GE-Healthcare and were diluted at 1/5000°. The Western Lightning Chemiluminescence (ECL) reagents were purchased from PerkinElmer.

Antibodies used in this study are listed in Supplementary Table 3. The horse-radish peroxidase (HRP)-conjugated anti-mouse (Anti mouse HRP linked antibody ref 7076 and Rabbit Anti-Mouse IgG (Light chain specific) mAb (HRP Conjugate) ref 58802) and rabbit IgG (Anti-Rabbit IgG HRP linked Antibody ref7074) antibodies were purchased from Cell Signalling Technology and were diluted at 1:5000 prior to use. HRP-conjugated anti-IgG α-goat (DAKO ref P0449) and anti-sheep (Sigma ref. A3415) were diluted 1:2500 for use. The Western Lightning Chemiluminescence (ECL) reagents were purchased from PerkinElmer. (Ref No. NEL104001EA).
Secondary, fluor conjugated anti-mouse and rabbit IgG antibodies were purchased from Thermo Fisher Scientific: Alexa fluor 555 A21424, Alexa fluor 444 A11029 and Alexa fluor 546 A11030. All antibodies were used at 1:1000 dilution.