Fkbp5 fkbp5

Lysates (8–15 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. 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). 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 1X TBST (Teknova, T9511). After blocking membranes were probed with the following primaries: FKBP5/FKBP5 (1:100; Cell Signaling Technology, 12,210), HTT (Millipore, MAB2166), polyQ 1C2 (1:500; Millipore, MAB1574), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441). Membranes were incubated with secondary anti-murine horseradish peroxidase (HRP)-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit HRP-coupled antibodies (1:2500; GE Healthcare, NA934 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 (8–15 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. 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). 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: FKBP5/FKBP5 (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 HRP-coupled antibodies (1:2500; GE Healthcare, NA934) 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.