Mouse α tubulin

Mouse α-GST (sc-138; 1:1,000), goat α-SRSF1 (sc-10254; 1:1,000), rabbit α-U2AF65 (sc-48804; 1:5,000), goat α-actin (sc-1616; 1:1,000), mouse α-tubulin (sc-8035; 1:3,000), goat α-BLM (sc-7790; 1:1,000), mouse α-SUMO1 (sc-5308; 1:1,000) and rabbit α-RNAPII (sc-899; 1:1,000) were purchased from Santa Cruz. Mouse α-RNAPII phospho-CTD (phospho S2; H5; ab24758; 1:5,000) and rabbit α-THOC5 (ab86070; 1:5,000) were purchased from Abcam. Rabbit α-Histone H4 (no. 2592; 1:1,000) and rabbit α-PIAS1 (no. 3550; 1:1,000) were purchased from Cell Signaling. Mouse α-NWSHPQFEK tag (StrepII tag; A01732; 1:3,000) was from GeneScript. Mouse α-p84 (GTX70220; 1:5,000) was from GeneTex and rabbit α-TOP1 (no. 3552–1; 1:5,000) was from Epitomics. Rabbit α-FLAG (F7425; 1:3,000) was purchased from Sigma-Aldrich. Mouse α-TOP1 (1:5,000) was kindly provided by Dr Yung-Chi Cheng (Yale School of Medicine). Mouse α-PCNA (1:5,000) was kindly provided by Dr Stephen West (Cancer Research UK). Rabbit α-RECQ5 (1:3,000) was generated as described^{15 (link)}. Mouse α-DNA-RNA hybrid [S9.6] was kindly provided by Dr Stephen H. Leppla National Institutes of Health (NIH).

Expression levels of WT and mutant POGLUT1 were examined by performing immunostaining in third instar wing imaginal discs overexpressing WT and mutant POGLUT1 driven by dpp-GAL4, which drives expression along the anterior-posterior boundary of the wing disc. Animals were raised at 25°C. UASattB-POGLUT1^WT-FLAG-VK22 and UASattB-POGLUT1^mutant-FLAG-VK22 animals were crossed with UAS-GFP; dpp-GAL4 animals to obtain UASattB-POGLUT1^WT-FLAG-VK22/ UAS-GFP; dpp-GAL4/+ or UASattB-POGLUT1^mutant-FLAG-VK22/ UAS-GFP; dpp-GAL4/+ animals. Staining was performed by α-FLAG antibody. The signal intensity of POGLUT1 for each imaginal disc was measured by ImageJ and normalized by the GFP signal intensity and the total area of the disc (also measured by ImageJ).
For Western blotting, proteins were extracted from wing imaginal discs in lysis buffer containing a protease inhibitor cocktail (Promega). The following antibodies were used: mouse α-FLAG M2 1:100 (Sigma), mouse α-tubulin 1:1000 (Santa Cruz Biotechnology Cat# sc-8035, RRID:AB_628408), goat α-mouse-HRP 1:2000 (Jackson Immuno Research Laboratories). Western blots were developed using Pierce ECL Western Blotting Substrates (Thermo Scientific). The bands were detected using an Image Quant LAS 4000 system from GE Healthcare.

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).