Anti fos

Protein quantification was performed using a BCA Kit. Protein lysates was subjected to SDS-PAGE and subsequently electro-transferred to a polyvinylidene fluoride membrane (Millipore). Western blotting was performed as previously described^{57 (link), 58 (link)}. The primary antibodies used were as follows (company, catalog number): anti-ATF6 (Santa Cruz, sc-22799), anti-BIP (Cell Signaling Technology, 3177), anti-P16 (BD Bioscience, 550834), anti-P21 (Cell Signaling Technology, 2947), anti-Lamin B, anti-LAP2 (BD Bioscience, 611000), anti-FOS (Cell Signaling Technology, 2250), anti-Calreticulin (Abcam, ab2907), anti-Calnexin (Cell Signaling Technology, 2679), anti-Ero1Lα (Cell Signaling Technology, 3264), anti-IRE1 (Cell Signaling Technology, 3294), anti-PDI (Cell Signaling Technology, 3501), anti-PERK (Cell Signaling Technology, 5683), anti-EMDM (Santa Cruz, sc-377394), anti-β-Actin (Santa Cruz, sc69879), anti-GAPDH (Santa Cruz, sc-25778), and anti-Flag (Sigma, F1804).

Immunoprecipitation of RIPK1 and MLKL was performed according to a previously described protocol [28] (link). Briefly, cells cultured in 10-cm dishes were lysed for 30 min on ice in lysis buffer containing 1% (w/v) Triton X-100, 0.15 M NaCl, 30 mM Tris-HCl (pH 7.5) and protease inhibitors (Roche, Germany). The lysates were sonicated and centrifuged at 10,000 g for 15 min at 4°C. One milligram of extracted protein in lysis buffer was incubated overnight with 10 µg rabbit anti-RIPK1 or anti-MLKL antibody followed by incubation with 20 µl Dynabeads Protein G (Invitrogen, USA) for 2 h at 4°C and three washes with lysis buffer. The beads were directly boiled in 1% SDS loading buffer for 5 min. Subcellular fractions, including nuclear, membrane and cytosolic fractions, were prepared using the proteoExtract sub-cellular proteome extraction kit (Calbiochem, USA) according to the manufacturer's instructions.
Western blot analysis was performed as described previously [27] (link), using the following antibodies: anti-caspase-9, anti-caspase-3, anti-Fos, anti-RIPK1, and anti-survivin antibodies from Cell Signaling Technology (Beverly, MA); anti-RIPK3 and anti-SMAC antibodies from Abcam (Cambridge, MA); anti-MLKL, anti-cytochrome C and anti-Bcl-2 antibodies from Santa Cruz Biotechnology.

For control experiments presented in Figure 1, mice were euthanized 3 weeks following viral delivery. Hit sites were verified using a marker virus (AAV-CMV-Cas9-HA(18)).
Upon the completion of DREADD studies, mice were injected with CNO (1 mg/kg), sacrificed 2 h post-injection, and then perfused with 10% formalin. Brains were then removed and post-fixed in 10% formalin for 24 hr, before being moved to 30% sucrose for 24 hr. Brains were then sectioned as 30 μm thick free-floating sections. Immunohistochemical and immunofluorescent staining was performed using standard procedures using anti-FOS (1:1000, #2250, Cell Signaling Technology), GFP (1:1000, #1020, Aves Laboratories), and DSRed (1:1000, #632392, Clontech) antibodies. Images were collected on an Olympus BX51 microscope.