Atxn2l

For Western blot, antibodies of ATXN2L (Proteintech, Rosemont, IL), pAKT (Cell Signaling Tech., Danvers, MA, US), and GAPDH (Proteintech) were used. Procedures was as previously described^{21 (link)}. Generally, cells or tissues were solubilized in lysis buffer (KeyGEN, Nanjing, China), and total protein concentration was determined by BioRad BCA method (Pierce, Rockford, IL, USA). After electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels, protein was electroblotted onto polyvinylidene fluoride membranes. Then, membranes were blocked with 5% bovine serum albumin and incubated overnight at 4 °C with antibodies against ATXN2L, pAKT, or GAPDH, respectively. After incubated by secondary antibodies, blots were visualized using enhanced chemiluminescence.

Protein was isolated with RIPA buffer as described before [106 (link)]. Samples with 20 µg protein were separated on 8% SDS gels and blotted on nitrocellulose membranes. Membranes were incubated overnight at 4 °C with the following antibodies: ATXN2L (Bethyl A301-369), ATXN2L (Invitrogen PA5-59601), ATXN2L (Proteintech 24822-1-AP), ATXN2 (Proteintech 21776-1-AP) or beta-Actin (Sigma A5441), and for 1 h with the respective secondary antibodies (Li-Cor). Fluorescence was detected with the Li-Cor Odyssey Classic Instrument and bands were analyzed with Image Studio Lite, Version 5.2.