Anti eef1bα

Westerns were carried out using antibodies and techniques as previously described [32] (link), with the following antibodies: anti-eEF1Bα from Proteintech (Manchester, UK; 1/2000) or Abcam (Cambridge, UK; 1/400), anti-eEF1Bδ from Proteintech Group (Manchester, UK; 1/3000), anti-eEF1Bγ from Abnova (Taipei, Taiwan; 1/2000) or Abcam (Cambridge, UK; 1/1000) and anti-GAPDH from Chemicon (Merck Millipore, Watford, UK; 1∶30,000). Calbiochem (Merck Millipore, Watford, UK) Rapid Step ECL was used for detection.

Slides of human tissues were obtained from Biochain (AMS Biotechnology, Abingdon, UK). Paraffin embedded sections of human and mouse tissues were deparaffinised, blocked in peroxidise blocking solution for 5 minutes and then washed and blocked in goat serum diluted 1∶5 with PBS for 10 minutes. Primary antibody was added as follows: anti-eEF1Bα from Proteintech (Manchester, UK; 1/100), anti-eEF1Bδ from Proteintech Group (Manchester, UK; 1/400) and anti-eEF1Bγ from Abnova (Taipei, Taiwan; 1/100). The slides were then incubated and visualised using ChemMate DAKO EnVision Detection Kit (DAKO) according to the manufacturer's instructions. In brief, the slides were then washed in PBS and three drops of ChemMate DAKO Envision/HRP Rabbit/Mouse secondary antibody (DAKO Cytomation; Agilent Technologies, Wokingham, UK) were added to each slide and incubated for a further 30 minutes. The slides were washed with PBS, removed from the sequenzer and 0.5 ml of DAB working solution was added to each slide and incubated for 2 minutes. Finally, the slides were washed in dH₂O, counterstained in haemotoxylin, stained with lithium carbonate and dehydrated in absolute ethanol and 75% ethanol, cleared in xylene and mounted in pertex. The entire procedure was performed at room temperature. Sections were viewed by light microscopy on Olympus BX51 using DP software (Olympus).