Nbt bcip

Samples from cells lysed in RIPA buffer containing protease/phosphatase inhibitors and benzonase, and cytoplasmic or nuclear fractions were resolved in a 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Denmark). Protein bands were detected with antibodies against ataxin-1 (SA4645), GFP (2956, Cell Signaling Technologies, USA), lamin A/C (4777, Cell Signaling Technologies, USA) and GAPDH (5171, Cell Signaling Technologies, USA) and visualized using either NBT/BCIP (AppliChem, Denmark) or Luminata Western HRP Chemiluminescence Substrate (Merck).

MSCs (5x10⁵ cells) were lysed in 200ul 1% SDS PBS. Cell extracts and supernatants were analyzed in SDS-PAGE electrophoresis followed by electrotransfer onto nitrocellulose membrane (Macherey-Nagel, Germany) using a semidry apparatus. Membranes were blocked with 5% w/v skim milk powder (Sigma Aldrich, USA) in PBST for 1h and incubated with mouse anti-GFP mAb (Millipore, MAB2510, 1:2000 dilution) or rabbit anti-TERT (Novus Biologicals, USA, NB120-32020, 1:2000 dilution) antibodies overnight. For loading controls, membranes were blotted with rabbit anti-GAPDH (Santa Cruz, USA, sc-25778, 1:1000 dilution) antibody. After incubation with the appropriate secondary alkaline phosphatase-conjugated antibody (Merck, Germany), membranes were developed with AP substrate NBT/BCIP (Applichem, Germany).