Temporal Profiling of TSHR during Adipogenesis

Cell lysates from OF at various time points before and during adipogenesis were obtained by addition of lysis buffer as previously described [13 (link)]. The culture supernatants from the same time points were also collected and concentrated using spin columns (Merck Millipore, Watford, Hertfordshire, UK) to produce an 80-fold concentration. Lysates and concentrated supernatants were separated using SDS-PAGE as previously described [13 (link)]. Briefly, proteins were extracted, at various time points, in Laemmli buffer containing 1 mM phenylmethylsulphonyl fluoride. Samples were separated by 10% SDS-PAGE and then the gel electroblotted onto PVDF membrane. The blots were probed using antibodies to the full length TSHR (2C11, Santa Cruz Biotechnology, Heidelberg, Germany) and TSHRv antibody, at dilutions of 1:200 and 1:50 respectively at 4 °C overnight. This was followed by a sheep anti-mouse IgG-HRP (1:5000, room temperature for 1 h, GE Healthcare) or donkey anti-rabbit IgG-HRP conjugate (1:5000, room temperature for 1 h, GE Healthcare) and then visualised by enhanced chemiluminescence using ECL Plus (Amersham Pharmacia Biotech, Buckinghamshire, UK). They were then stripped and reprobed with antibodies to housekeeping protein, actin at dilution of 1:1000 4 °C overnight with secondary anti-rabbit as above.