For gel electrophoresis, tissue samples were adjusted to a concentration of 0.8 μg/μl with sterile water, reducing buffer (Invitrogen), and denaturing buffer (Invitrogen). Samples were then incubated at 70°C for 10 min.
The Novex Mini Cell NuPAGE system (Invitrogen) with 4–12% Bis–Tris gradient polyacrylamide gels (Invitrogen) was used. A 8 μg of denatured protein homogenate was run in each lane. Samples were loaded in duplicate in adjacent lanes, and a molecular weight standard was run on each gel. A lane containing 8 μg of homogenized macaque cortex was also loaded onto each gel to control for interblot variability. Gels were suspended in a bath of NuPAGE MES SDS running buffer (Invitrogen) with 500 μl NuPAGE antioxidant (Invitrogen) during electrophoresis.
Following electrophoresis, proteins were transferred onto Immobilon-FL PVDF membranes (Millipore) using a semi-dry transfer apparatus (Bio-Rad). After electroblot transfer of the protein, membranes were washed twice and incubated with Odyssey Blocking Buffer (Li-Cor Biosciences) for 1 h at room temperature with rocking to block non-specific antibody binding. Membranes were exposed to the primary polyclonal antibody diluted 1:10,000 for actin (Chemicon MAB150R), VCP (Abcam ab11433), and β-tubulin (Upstate 05-661), and 1:20,000 for GAPDH (Sigma G9545) in blocking buffer with 0.1% tween overnight at 4°C with rocking. Next, the membranes were washed three times for 10 min in tris-buffered saline with 0.1% tween (TBST), then rocked for 1 h with anti-mouse IR-Dye 680 or 800 CW secondary antibody (Li-Cor Biosciences) diluted 1:10,000 in blocking buffer with 0.1% tween. Membranes were washed three times for 10 min in TBST then washed five times in high purity water and allowed to dry for 3–5 min before scanning (infrared imaging system; Li-Cor Biosciences). We pre-tested the β-tubulin, actin, GAPDH, and VCP Western blot assays using varying concentrations of protein from a human cortical tissue homogenate sample. These experiments demonstrated that each assay was linear with protein concentrations found in this study (VCP: R = 0.99, P < 0.01; β-tubulin: R = 0.99, P < 0.01; actin: R = 0.95, P = 0.01; GAPDH: R = 0.98, P < 0.01).
The Novex Mini Cell NuPAGE system (Invitrogen) with 4–12% Bis–Tris gradient polyacrylamide gels (Invitrogen) was used. A 8 μg of denatured protein homogenate was run in each lane. Samples were loaded in duplicate in adjacent lanes, and a molecular weight standard was run on each gel. A lane containing 8 μg of homogenized macaque cortex was also loaded onto each gel to control for interblot variability. Gels were suspended in a bath of NuPAGE MES SDS running buffer (Invitrogen) with 500 μl NuPAGE antioxidant (Invitrogen) during electrophoresis.
Following electrophoresis, proteins were transferred onto Immobilon-FL PVDF membranes (Millipore) using a semi-dry transfer apparatus (Bio-Rad). After electroblot transfer of the protein, membranes were washed twice and incubated with Odyssey Blocking Buffer (Li-Cor Biosciences) for 1 h at room temperature with rocking to block non-specific antibody binding. Membranes were exposed to the primary polyclonal antibody diluted 1:10,000 for actin (Chemicon MAB150R), VCP (Abcam ab11433), and β-tubulin (Upstate 05-661), and 1:20,000 for GAPDH (Sigma G9545) in blocking buffer with 0.1% tween overnight at 4°C with rocking. Next, the membranes were washed three times for 10 min in tris-buffered saline with 0.1% tween (TBST), then rocked for 1 h with anti-mouse IR-Dye 680 or 800 CW secondary antibody (Li-Cor Biosciences) diluted 1:10,000 in blocking buffer with 0.1% tween. Membranes were washed three times for 10 min in TBST then washed five times in high purity water and allowed to dry for 3–5 min before scanning (infrared imaging system; Li-Cor Biosciences). We pre-tested the β-tubulin, actin, GAPDH, and VCP Western blot assays using varying concentrations of protein from a human cortical tissue homogenate sample. These experiments demonstrated that each assay was linear with protein concentrations found in this study (VCP: R = 0.99, P < 0.01; β-tubulin: R = 0.99, P < 0.01; actin: R = 0.95, P = 0.01; GAPDH: R = 0.98, P < 0.01).
