β tubulin antibody

SW982 human synovial sarcoma cells were cultured in Roswell Park Memorial Institute (RPMI 1640; Gibco) medium supplemented with 10% HyClone^TM FBS (GE Healthcare) and 1% penicillin-streptomycin (Gibco), at 37°C with 5% CO₂. The cells were seeded in 6-well plates at least 6 h prior to transfection using the ViaFectTM transfection reagent (Promega). One day after transfection, cells were harvested and lysed. Total cell lysates were resolved on a denaturing 10% SDS-PAGE gel, and blotted onto a PVDF membrane using the Trans-Blot Turbo Transfer system (Bio-Rad). Samples were then probed with (1) Nanoluc antibody (R&D Systems; MAB100261), resuspended to 306 μg/mL and diluted to 1:200 with 5% milk in TBST, and (2) β-tubulin antibody (Cell Signaling, # 2146), which was supplied in a concentration of 100 μg/mL, and diluted to 1:1000 with the 5% bovine serum albumin (BSA) in TBST. The samples were then probed with the appropriate secondary antibody: (1) α-mouse secondary antibody diluted to 1:2500 in blocking solution for Nanoluc, and (2) α-rabbit secondary antibody diluted to 1:2500 in blocking solution for β-tubulin. The membrane was incubated in substrate ECL HRP substrate (Advansta), and chemiluminescent signals were obtained using the ImageQuant LAS 4000 mini (GE Healthcare Life Sciences).

Subcellular fractions were prepared from groups of five 10–15-week-old and 30-50-week-old wt and cTnI-G203S hearts, an NE-PER^TM Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific, 78833), supplemented with cOmplete^™, Mini, EDTA-free Protease (Roche, 4693159001) and PhosStop^TM phosphatase (Roche, 4906837001) inhibitor tablets, according to manufacturer protocols. Blots were probed with the following primary antibodies: Total mTOR, mTOR (7C10) rabbit mAb (Cell Signaling Technology, 2983, 1:1000); Active mTOR, phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (Cell Signaling Technology, 5536, 1:1000); Total SRP6, S6 ribosomal protein (5G10) rabbit mAb (Cell Signaling Technology, 2217, 1:1000); Active SRP6, phospho-S6 ribosomal protein (Ser235/236) (2F9) rabbit mAb (Cell Signaling Technology, 4856, 1:1000). The following primary antibodies were used for loading controls: β-tubulin antibody (Cell Signaling Technology, 2146, 1:500); histone H2B (D2H6) rabbit mAb (Cell Signaling Technology, 12364, 1:1000). Blots were probed with goat anti-rabbit IgG H&L (HRP) preadsorbed secondary antibody (Abcam, ab97080, 1:10000). The same antibodies were used to confirm purity of cytoplasmic and nuclear fractions (Supplementary Fig. 5).

Cells were seeded for 72 h on a chamber slide (Thermo Fisher Scientific, Waltham, MA, USA) and conjugated with OBP-301, PTX, or both for 24 h. After each treatment, cells were fixed for 15 min in 4% formaldehyde at room temperature. Protein blocking was done with 5% bovine serum albumin-PBS for 10 min at room temperature followed by incubation with β-tubulin antibody (Cell Signaling Technology, Danvers, MA, USA) for 24 h at 4°C. Alexa Fluor 488 anti-rabbit secondary antibody (Thermo Fisher Scientific) was then applied for 60 min at room temperature. Nuclear morphology was counterstained by using VECTASHIELD Hardset Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The morphological changes of the nuclei and cytoskeleton were examined under a LSM780 confocal laser scanning microscope (Zeiss). Images were processed with Imaris 7.6 (Bitplane, Belfast, UK).