Ecl prime western blot detection kit

Western blot analysis was conducted as previously described [66 (link), 67 (link)]. Briefly, whole-cell lysates were extracted in urea lysis buffer (8.8urea, 5NaH₂PO₄, 1Tris, pH 8.0) and stored at −80°C for future use. A total of 80 μg of protein was mixed with 6x Lamelli buffer and boiled for 10 minutes. Protein extracts were run on a 4-15% pre-cast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) for 1 hour at a constant voltage of 150 V. Proteins were then transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA) for 1 hour at a constant current of 350 mA. The primary antibodies were as follows: monoclonal anti-BRG1 antibody (sc-17796, 1:200, Santa Cruz Biotechnology); monoclonal mouse anti-p21 (556430, 1:500, BD Pharmingen, San Jose, CA, USA); polyclonal rabbit anti-p16 (10883-1-AP, 1:500, Protein Tech, Chicago, IL, USA). Glyceraldehyde 3-phosphate dehydrogenase antibody (GeneTex Inc., Irvine, CA, USA) was used as the loading control. Anti-mouse and anti-rabbit secondary antibodies were purchased from GE Healthcare (Buckinghamshire, England, UK). Western blots were developed using an ECL Prime Western blot detection kit (GE Healthcare) and were analyzed with ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).

Cell pellets were thawed on ice, lysed by triturating in PBS containing 0.05% Triton-X and a cOmplete mini protease inhibitor tablet (Roche), and clarified by 5 min sequential centrifugations at 500 x g and 1000 x g. Total protein concentration of the clarified lysate was measured using Bradford Assay (Bio-Rad). Clarified lysate was mixed with 2X SDS buffer (final SDS concentration 4%) and run on NuPAGE 4–12% Bis-Tris Gel at 150V for ~75 min. The gel was then transferred onto Immobilon P membrane for 1 hr at 20V using a semi-dry transfer apparatus (Bio-Rad). The membrane was then blocked with 5% non-fat dry milk in TBST for 1 hr before primary rabbit anti-tau monoclonal antibody (Tau A, which was raised against QTAP…KIGSTENL) was added at 1:1000 and placed in a shaker overnight at 4°C. The membrane was then washed four times with TBST at 10 min intervals. The membrane was then re-probed with goat anti-rabbit secondary antibody for 1.5 hr at room temperature. The membrane was then washed four times with TBST. Finally, the membrane was exposed to ECL Prime western blot detection kit (GE Lifesciences) for 2 min and imaged with a Syngene digital imager. Images are representative of at least three similar replicates.

For Western blot and dot blot analyses, immunoprecipitation was eliminated to maximize the quantity of tau protein and enhance tau visibility on the blots. SEC fractions were boiled for 5 min with SDS-PAGE sample buffer and loaded into a NuPAGE 4% to 12% Bis-Tris Gel in a chamber filled with NuPAGE MOPS SDS running buffer (ThermoFisher) and run at 100 V for ∼110 min. Samples were then transferred to a PVDF membrane using a semidry transfer apparatus (Bio-Rad). After being blocked in 5% milk (Bio-Rad), the membrane was incubated with primary polyclonal rabbit anti-human tau antibody (Dako, Agilent) at 1:2000 on a shaker overnight at 4 °C. It was then washed with TBST 3 times for 30 min and was incubated with anti-rabbit secondary antibody for 1 h at room temperature. The membrane was then washed with TBST on a shaker for 10 min twice, before the final rinse with TBS. The membrane was then developed with ECL prime Western blot detection kit (GE Lifescience) for 3 min before being imaged by a digital imager (Syngene).