Phospho rps6ser240 244

The whole-cell protein extracts were prepared using RIPA buffer with protease and phosphatase inhibitor cocktails (Thermo Scientific, Thermo Fisher Scientific Inc., Waltham, MA, USA), resolved by SDS–PAGE on 4–20% gels and transferred to Odyssey nitrocellulose membranes (LI-COR Biosciences, Lincoln, NE, USA). Membranes were blocked with Odyssey Blocking Buffer and incubated with primary antibodies overnight at 4°C, followed by IRDye® secondary antibodies (LI-COR Biosciences). LI-COR Odyssey Imager was used for the band visualization. Equal loading and adequate transfer to the membranes were verified by staining with Ponceau S (Sigma-Aldrich) and with anti-GAPDH (Sigma-Aldrich) antibody. We used Abs against: REDD1 (Proteintech Group, Inc., Chicago, IL), LC3B, phospho-rpS6^Ser240/244, phospho-4E-BP1^Thr37/46, phospho-GR^Ser211 (Cell Signaling Technology, Inc.), and GR (H-300 or M-20, Santa Cruz Biotechnology, Inc.).
Abs against REDD1 and Beclin-1 (Fig2B and C) were reported to recognize multiband pattern on Western blots (Katiyar et al, 2009 (link); Li et al, 2012 (link); Regazzetti et al, 2012 (link), and http://www.cellsignal.com/products/primary-antibodies/3738?Ntt=beclin&fromPage=plp), which may reflect phosphorylation status of these proteins.

Western blot experiments were performed as described previously [26 (link)]. Sample lysate protein concentrations were normalized with lysis buffer and denatured with 4 × Laemmli buffer with 10% β-mercaptoethanol. Proteins were resolved in 10% Tris–Glycine gels (BioRad) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010). Membranes were blocked in 1X PBS with 0.1% Tween-20 (PBS-T). All antibodies were diluted in 5% milk or 5% BSA in PBS-T. Primary antibodies were used as follows: PHF1 (1:2000, generously provided by Dr. Peter Davies), H150 total tau (1:2000, SantaCruz), Tau 5 total tau (1:2000, Millipore), actin (1:5000, Cell Signaling Technology), GAPDH (1:5000, Cell Signaling Technology), RPL28 (1:1000, GeneTex), EIF3E (1:1000, Sigma-Aldrich), Phospho-RPS6 Ser240/244 (1:1000, Cell Signaling Technology), total RPS6 (1:1000, SantaCruz). Bands were detected using ECL (GE Amersham Imager 600) using SuperSignal West Pico (Thermo Fisher, 1863096). Blot images were quantified using ImageJ (1.52b) and normalized to either GAPDH or β-actin.

Whole cell protein extracts were prepared as described [27 (link)]. Separate cytoplasmic and nuclear protein fractions were isolated with NE-PER Nuclear and Cytoplasmic Extraction Kit according to manufacturer protocol (ThermoFisher Scientific, Waltham, MA). The proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (LI-COR Biosciences; Lincoln, NE). After blocking, membranes were incubated with primary antibodies overnight at 4°C, followed by IRDye® secondary antibodies (LI-COR Biosciences). LI-COR Odyssey Imager was used for the band visualization. The antibodies against FKBP51, GR, RelA/p65, p65^Ser536 (Santa Cruz Biotechnology, Dallas, TX), Redd1 (Proteintech; Chicago, IL), phospho-GR^Ser211, Akt, phospho-Akt^Ser473, phospho-rpS6^Ser240/244, phospho-p70/S6K^Thr389, lamin B, tubulin (Cell Signaling, Danvers, MA), and GAPDH (Sigma, St Louis, MO) were used at concentrations recommended by their manufacturers. The multi-band pattern of FKBP51 signal on Western blots (Figure 1a) may reflect FKBP51 post-translational modifications such as phosphorylation [41 (link)].
ImageJ (http://rsb.info.nih.gov/ij/index.html) was used for densitometry. The intensity of bands was normalized to the corresponding loading control and expressed as fold of change vs vehicle-treated WT or Cas9-only control.