Mouse α gfp

Flp-In T-Rx 293 cell lines expressing GFP-0N4R tau constructs were generated, propagated, and stored according to the manufacturer’s instructions (https://www.thermofisher.com/order/catalog/product/R78007). 150K cells were seeded in a 6-well plate in 3 mL complete growth medium (DMEM, 10% FBS, 1% Penicillin/Streptomycin), allowed to adhere for 12h, and induced with 10 μg/mL Doxycycline for 72h. Media was removed and cells were washed with DPBS. Cell membranes were lysed with 90 μL of a microtubule-stabilizing lysis buffer (20 mM MOPS pH 6.8, 50 mM NaCL, .5% NP-40, 2 mM EGTA, 1 mM MgCl₂, 2 mM TCEP, 2 μg/mL paclitaxel, 2 mM GTP, 250 unit/mL benzonase) and rocked gently for 3 min. Soluble lysate was removed gently with a cut pipette tip. The well was rinsed gently with 45 μL of lysis buffer and the soluble fraction and centrifuged at 15K RPM at room temp. 50 μL of was diluted in 3X SDS-PAGE loading buffer. 200 μL of 1X SDS-PAGE loading buffer was added to the well to solubilize the microtubule fraction. Samples were boiled and 15 μL loaded for Western Blot analysis. Membranes were probed with mouse αGFP (Santa Cruz), mouse αTubulin (Santa Cruz), and Rabbit αCHIP (Abcam). Blots were developed using Licor secondary antibodies (Goat-αRabbit 680RD and Goat-αmouse 800CW).