Phospho grser211

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.

Hippocampal tissue proteins isolation and immunoblots were performed as previously described (Cohen et al., 2016). Antibodies against GR (Santa Cruz Biotechnology Inc, Cat# sc‐8992), phospho‐GR Ser 211 (Cell Signaling, Cat# 4161), FKBP4 (Cell Signaling, Cat# 11826S) and FKBP5 (Santa Cruz Biotechnology Inc., Cat#sc‐13983) and at 1:50 dilution were used to determine protein abundance and Vinculin (Cell Signaling, Cat #13901) at 1:10,000 dilution was used as a loading control.

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.