Fkbp5 fkbp51

Cortical and striatal lysates (20–40 μg) were prepared in 4X LDS buffer (Invitrogen, NP0007) and 0.05 M DTT. Samples were boiled for 10 min at 95°C and separated by 1D-SDS-PAGE electrophoresis on a 12% Bis-Tris gel at 200 V for 50 min in MOPS running buffer and antioxidant (Invitrogen, NP0005). Transfer was performed on ice in a cold room at 350 mA constant for 1 h in transfer buffer (Invitrogen, NP00061) with 10% methanol onto a 0.2-μm PVDF membrane (Thermo Fisher, 88,520). After blocking in 5% milk in TBST, primary antibody FKBP1A/FKBP12 (1:1000–4000; Abcam, ab2918), FKBP1B/FKBP12.6 (1:100–200, Abcam, ab82316); FKBP5/FKBP51 (1:100–500; Cell Signaling Technology, D5G2), FKBP4/FKBP52 (1:100; Cell Signaling Technology, 11,826), TUBA4A/TUBA1A/α-tubulin (1:500–4000; MilliporeSigma, T6199) were incubated ON in either 5% milk TBST or 5% BSA TBST at 4°C. Membranes were incubated with secondary anti-rabbit (1:2500; GE Healthcare NA934) or anti-murine HRP-coupled antibodies (1:2500; GE Healthcare, NXA931) at RT for 2 h in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.

Lysates (15–30 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. The R6/2 lysates were run on a 4–12% Bis-tris gel at 200 V for 50 min in MES running buffer (Invitrogen, NP0002) with antioxidant (Invitrogen, NP0005). The zQ175 lysates were separated on 3–8% tris-acetate gel at 200 V for 90 min in tris-acetate running buffer. Transfer was performed ON, 20 V for 840 min at 4°C, onto a 0.4-μm nitrocellulose membrane. Blocking was done with 5% milk in TBST. After blocking membranes were probed with the following primaries: HTT (1:500; MilliporeSigma, 5492), FKBP5/FKBP51 (1:100; Cell Signaling Technology, 12,210), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441), or TUBA4A/TUBA1A/α-tubulin (1:1000; MilliporeSigma,T6199). Membranes were incubated with secondary anti-murine HRP-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit (1:2500;GE Healthcare, NA934) at RT for 2 h or ON at 4°C in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.