Anti β tubulin antibody

Cell culture reagents were purchased from Gibco Laboratories (Grand Island, NY, USA). The SAF32 anti-PrP antibody was from Cayman Chemical (Ann Arbor, MI, USA). The recombinant His-tagged 37LRP polypeptide (r37LRP) was made in bacteria and Nickel affinity purified, as previously described [27 (link)]. The polyclonal 4290 Ab was made against a C-terminal peptide derived from LR and was a kind gift from Dr. Mark E. Sobel (Bethesda, MD, USA); NSC48478 inhibitor has been already described [27 (link)]. Protein-A-Sepharose was from Pharmacia Diagnostics AB (Uppsala, Sweden). Transferrin Alexa-594- conjugated (Tfr), Alexa-488-, Alexa-546-conjugated secondary Abs and LysoTracker Red DND-99 were from Invitrogen (Molecular Probes). The anti- KDEL, anti-Giantin, antibodies were from StressGen Biotechnologies Corp (Victoria, BC, Canada). Anti-β-tubulin antibody was from Abcam. Anti-phospho RPS6 antibody (Ser240/244), anti-GSK and anti-pGSK3β Ser9 were from Cell Signalling Technology. DAPI was purchased from Cell Signal Technology. Biotin-LC was from Pierce and all other reagents were from Sigma Chemical Co. (St Louis, MO, USA). NSC48478 [1-((4-chloroanilino)methyl)-2-naphtol] was obtained from the NCI/DTP Open Chemical repository (http://dtp.cancer.gov), dissolved in DMSO and stored at −20 °C.

Cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail) for 30 min on ice. Lysates were centrifuged at 2,0000 g for 30 min at 4 °C. The supernatant was mixed with SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, and 20% glycerol with bromophenol blue) and heated for 5 min. Proteins were separated by 12% SDS-PAGE gel and transferred to PVDF membranes. The membrane was blocked in 5% non-fat milk, and incubated with the intended primary antibody in TBS containing 0.1% Tween 20 (TBS-T) for 3 h. After washing with TBST-T, the membrane was incubated with HRP-conjugated secondary antibody for 1 h. After three washes with TBST-T, bands were visualized by chemiluminescence. The primary antibodies used in this study were: as follow anti-transgelin-2 (Novus Biologicals, CO, USA), anti-β-actin (Cell Signaling Technology, MA, USA), anti-Flag (Sigma-Aldrich, MO, USA), anti-GFP (Abcam, MA, USA), anti-ERK1/2 (Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2-T202/y204 antibody(Cell Signaling Technology, MA, USA), anti-β-tubulin antibody (Abcam, MA, USA), anti-GST antibody (Abcam, MA, USA).

The quantitated cell lysates were separated on 8%–12% SDS polyacrylamide gels and transferred onto PVDF membranes. After blocking for 1 h, the membranes were incubated sequentially with anti-V5 antibody (Life technologies, Carlsbad, CA), anti-β-actin antibody (Abcam, Cambridge, MA), anti-CDH1 antibody (BD Biosciences, San Diego, CA), anti-VIM antibody, anti-β-catenin ( Millipore, Temecula, CA), anti-CDH2 antibody (BD Bioscience, San Diego, CA), anti-SNAI2 antibody, anti-c-JUN antibody (Santa Cruz Biotechnology, Dallas, TX), and anti-β-tubulin antibody (Abcam, Cambridge, MA) in PBST. After washing for 3 times, the bound antibody was detected using the Enhanced Chemiluminescence System.

Human multiple myeloma cell lines (RPMI8226, U266) were obtained from Dr. Slavica Vuckovic (QIMR Berghofer Medical Research Institute). Human peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy volunteers and were collected under the ethical approval BPS/08/14/HREC. The cells were cultured in RPMI-1640 medium (Gibco, VIC, Australia) containing 10% (V/V) fetal bovine serum (FBS) (Gibco), 200 mM L-glutamine (Invitrogen, VIC, Australia), and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The Trx1 antibody (5G8) is a mouse monoclonal antibody generated against recombinant human thioredoxin [62 (link)]. The monoclonal anti-TrxR1 antibody was purchased from R&D systems (MN, USA). The polyclonal anti-NF-кβ p65 antibody was purchased from GeneTex (CA, USA) and anti-β-tubulin antibody was purchased from Abcam. The Trx1 inhibitor PX-12 (1-methylpropyl 2-imidazolyl disulfide) was purchased from Cayman Chemicals (MI, USA). The NF-κβ inhibitors BAY 11-7082 and curcumin were purchased from Cayman Chemicals and Sigma. The TrxR1 validated small interfering RNA (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology (TX, USA). The Trx1-anti sense plasmid was made by reversing the orientation of the Trx insert in pcDNA3.1 (Invitrogen) of a pTrx-WT plasmid previously described [63 (link)].

Cell culture reagents were purchased from Gibco Laboratories (Grand Island, NY, USA). Transferrin Alexa-594- conjugated (Tfr), cy2-, cy5-conjugated secondary Abs and Mitotracker Red CMXRos were from Invitrogen (Molecular Probes, Eugene, OR, USA). The anti-Giantin antibody was from StressGen Biotechnologies Corp (Victoria, BC, Canada). Anti-β-tubulin antibody was from Abcam. Anti-GSK, anti-pGSK3β Ser9 and DAPI were from Cell Signalling Technology. Anti-APP rabbit polyclonal antibody A8717 was from Sigma-Aldrich (St. Louis, MO, USA). Anti-Aβ antibody 6E10 (previously Covance, SIG-39320) and 4G8 were from Biolegend. All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA). NSC47924 [1-((4-methoxyanilino)methyl)-2-naphtol] was obtained from the NCI/DTP Open Chemical repository (http://dtp.cancer.gov), dissolved in DMSO and stored at −20 °C.

Proteinase K (PK), 1-hydroxybenzotriazole, N, N′-diisopropylcarbodiimide, trifluoroacetic acid, triisobutylsilane, piperidine, and dichloromethane were obtained from Sigma Chemical Co. (St. Louis, MO). Peptide N-glycosidase F (PNGase F) and protease inhibitor cocktail were obtained from New England Biolabs (Beverly, MA). Fmoc-amino acids and amino-PEG cellulose membranes were obtained from Intavis (San Marcos, CA). N-α-Fmoc-O-benzyl-L-phosphothreonine and N-α-Fmoc-O-benzyl-L-phosphoserine were purchased from AnaSpec (San Jose, CA). ECL Plus for enhanced chemiluminescence was purchased from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). The following anti-PrP antibodies were used: 3F4 directed against human PrP residues 106–110 [16 (link)], 1E4 against human PrP97–105 [17 (link), 18 (link)] (Cell Sciences, Inc., Canton, MA), and anti-C against PrP220–231 [17 (link), 18 (link)]. Anti β-tubulin antibody was purchased from Abcam (Cambridge, MA).