Anti 5lo

RIPA extracts from mouse brain homogenates were used for western Blot analyses as previously described^{14 (link),15 (link)}. Briefly, samples were electrophoresed on 10% Bis-Tris gels or 3–8% Tris-acetate gel (Bio-Rad, Richmond, CA), transferred onto nitrocellulose membranes (Bio-Rad) and then incubated overnight at 4 °C with the appropriate primary antibodies; anti-5LO [dilution: 1:200] (Santa Cruz, Dallas, TX), anti-HT7 [1:200] (Thermo, Waltham, MA), anti-AT8 [1:100] (Thermo), anti-AT270 [1:200] (Thermo), anti-PHF13 [1:100 (Thermo)], anti-SYP [1:300] (Santa Cruz), anti-PSD95 [1:200] (Thermo), anti-GSK3α/β [1:100] (Cell Signaling, Danvers, MA), anti-pGSK3α/β [1:100] (Cell Signaling), anti-SAPK/JNK [1:100] (Cell Signaling), anti-pSAPKJNK [1:100] (Cell Signaling), anti-cdk5 1[:200] (Santa Cruz), anti-p35/p25 [1:100] (Santa Cruz), anti-PP2A [1:200] (Santa Cruz), anti-GFAP (Santa Cruz), anti-Iba1[1:100] (Thermo) and anti-Beta actin [1:500] (Santa Cruz). After three washings with T-TBS (pH 7.4), membranes were incubated with IRDye 800CW-labeled secondary antibodies (LI-COR Bioscience, Lincoln, NE) at room temperature for 1 h. Signals were developed with Odyssey Infrared Imaging Systems (LI-COR Bioscience, Lincoln, NE). β-Actin was always used as an internal loading control.

RIPA (radio immunoassay precipitation) extracts from mouse brain homogenates were used for Western blot analyses as previously described (Di Meco et al., 2014; Giannopoulos et al., 2013). Briefly, samples were electrophoresed on 10% Bis–Tris gels or 3%–8% Tris–acetate gel (Bio‐Rad, Richmond, CA, USA), transferred onto nitrocellulose membranes (Bio‐Rad), and then incubated overnight at 4°C with the appropriate primary antibodies; anti‐5LO [dilution: 1:200] (Santa Cruz, Dallas, TX, USA), anti‐HT7 [1:200] (Thermo, Waltham, MA, USA), anti‐AT8 [1:100] (Thermo), anti‐AT180 [1:200] (Thermo); anti‐AT270 [1:200] (Thermo), anti‐PHF1 (generous gift of Dr. Peter Davies); anti‐PHF13 [1:100 (Thermo)], anti‐SYP [1:300] (Santa Cruz), anti‐PSD95 [1:200] (Thermo), anti‐MAP2 [1:1,000] (Millipore), anti‐GSK3α/β [1:100] (Cell Signaling, Danvers, MA, USA), anti‐pGSK3α/β [1:100] (Cell Signaling), anti‐cdk5 [1:200] (Santa Cruz), anti‐p35/p25 [1:100] (Santa Cruz), anti‐GFAP (Santa Cruz), anti‐CD45 [1:100] (Thermo) and anti‐Beta actin [1:500] (Santa Cruz). After three washings with T‐TBS (pH7.4), membranes were incubated with IRDye 800CW‐labeled secondary antibodies (LI‐COR Bioscience, Lincoln, NE, USA) at room temperature for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). β‐Actin was always used as internal loading control.