Antibody against tubulin t5168

Jurkat cells were treated with 0, 0.25, 0.5, 1, 2 or 4 μg/ml D112 for 24 h. All cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1mM β-mercaptoethanol). The whole cell lysates were then mixed with 6X SDS-PAGE gel loading buffer and boiled for 10 min. Protein concentration was measured using Pierce BCA protein assay kit from Thermo Fisher Scientific Inc (Waltham, MA). 15 μg proteins from cell lysates were loaded onto 14% SDS polyacrylamide gels and subjected to SDS-PAGE analysis. Proteins were resolved at 100 V and subsequently transferred to PVDF membranes by use of Bio-Rad Mini-Gel Box Electrotransfer for 1 h at 300 mA. Membranes were blocked in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 5% skimmed milk for 1 h. Proteins were visualized with specific primary antibodies, followed by goat anti—mouse or rabbit HRP-conjugated secondary antibodies.
Antibody against caspase 3 (ADI-AAP-13) was from Enzo Life Science (Farmingdale, NY, USA); Caspase 9 antibody (9502), Bax antibody (2774) and Bcl-2 (2872) antibody were from Cell Signaling (Boston, MA, USA); Bak antibody (06–536) was from Thermo Fisher Scientific; Tom-20 antibody (SC-11415) was from Santa Cruz (Dallas, TX); Cytochrome c antibody (556433) was from BD Pharmingen (San Diego, CA) and antibody against Tubulin (T5168) was from Sigma-Aldrich.

Antibodies against p-RelA-S536 (#3033; 1:1000), RelA (#8242; 1:1000), RelB (#4922, 1:2000; IHC: #10544), c-Rel (#4727; 1:1000), NF-κB2 (#3017; 1:1000), LKB1 (#3050; 1:1000), and NUAK1 (#4458; 1:1000) were purchased from Cell Signalling Technology (Danvers, MA). Antibodies against lamin A/C (MAB3211; 1:500) and actin (A2066; 1:20000) was purchased from Millipore (Temecula, CA, USA). Antibody against tubulin (T5168; 1:20000) was purchased from Sigma. HRP-conjugated antibodies against mouse IgG (NA931; 1:10000) and rabbit IgG (NA934; 1:10000) were purchased from Cytiva. All antibodies were diluted in tris-buffered saline-Tween 20 containing 5% bovine serum albumin, with the exception of NUAK1 (tris-buffered saline-Tween 20 containing 5% nonfat milk). BAY 11-7082 was purchased from Cayman Chemical (cat. 10010266; Ann Arbor, MI, USA). ROS-Glo H₂O₂ Assay was purchased from Promega (cat. G8820).