Anti β tubulin antibody

Forty-eight hours post transfection cellular lysates or cellular medium were collected for experiments. Cells were lysed in equal volumes of pre-heated 2xSDS loading buffer (Sodium Dodecyl Sulphate 125 mM, Tris-HCl pH 6.8, 20% Glycerol, 2% SDS, 2% β-mercaptoethanol and bromophenol blue) and homogenized via sonication. Antibodies used against the ER-Stress proteins were: anti-BiP, anti-PERK, anti-P-PERK (Cell Signaling Technology, Danvers, MA) and anti-CHOP, anti-p-eIF2a (SantaCruz Biotechnology, CA), followed by peroxidase-labelled secondary antibodies either goat anti-mouse or donkey anti-rabbit (SantaCruz Biotechnology, CA). Proteins were detected using the Enhanced ChemiLuminescence (ECL) Plus Blotting Detection system (Amersham Biosciences, Buckinghamshire, UK) and were visualized by autoradiography on photographic film (KODAK X-OMAT, NY). All transblots were reprobed with anti-β-tubulin antibody (SantaCruz Biotechnology, CA) to prove equal amounts of protein were loaded on the membrane. Band density was defined using the ImageJ Software (http://imagej.nih.gov/ij).

Anti-gremlin-1 antibody was from Origene (TA324077), anti-E-cadherin antibody from BD Biosciences (610182) and anti-β-tubulin antibody from Santa Cruz (sc-9104). Integrin alpha v blocking antibody was from Millipore (MABT207) and used at 10 μg/ml concentration. Antibodies used for immunohistochemistry were anti-human calretinin (Abcam, ab16694), anti-mouse CD31 (Abcam, ab124432), anti-Ki67 (Abcam, ab16667) and anti-human laminA+C nuclear envelope marker (Abcam, ab108595), which was used to identify human tumor cells in mouse tissue. Broad spectrum MMP inhibitors GM6001 and BB2516 were from Calbiochem and used at 10 μM concentration. TGF-βR inhibitors SB431542 and SB505124 were from Sigma-Aldrich and used at 10 μM concentration. BMP receptor inhibitor LDN193189 was from Sigma-Aldrich and used at 100 nM concentration. BMP-2 was from R&D Systems and used at 25 ng/ml concentration. The recombinant fusion protein containing the ectodomain of human ActR2B (sActR2B-Fc) or anti-Müllerian hormone receptor (sAMHR2-Fc) fused to the Fc domain of human IgG1 and recombinant follistatin were produced as described previously [40 (link)] and used at 10 μg/ml. Soluble receptors sequester ligands and inhibit their binding to cell surface receptors.

LPS, sodium nitrite, phenylmethylsulfonyl fluoride, NP40 cell lysis buffer, dimethyl sulfoxide, protease inhibitor cocktail, and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). The EZ-Cytox cell viability assay kit was purchased from DAEIL lab (Seoul, Korea). The RNeasy kit was purchased from QIAGEN (Valencia, CA, USA). The PrimeScript II 1st Strand cDNA Synthesis Kit and SYBR premix were purchased from Takara Bio Inc. (Tokyo, Japan). Anti-iNOS and -STAT1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), anti-β-tubulin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6, TNF-α, and monocyte chemoattractant protein (MCP-1) were purchased from R&D Systems (Minneapolis, MN, USA), and the ELISA kit for IFN-β was purchased from Pestka Biomedical Laboratories (Piscataway, NJ, USA).

48 h after siRNA transfection, the cells were washed with PBS buffer and lysed with RIPA buffer (1× PBS, 1% IP-40, 0.5% sodium deoxycholate, and 0.1% SDS). After 1 h incubation on ice, the cellular mixture was centrifuged, and the supernatant was collected. Equivalent amounts (∼30 μg) of prepared cellular extracts were separated on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad). The membranes were probed with antibodies against human Polλ (Bethyl Lab) or Flag (Sigma-Aldrich), followed by appropriate secondary antibodies conjugated with horseradish peroxidase. The signals were detected using ECL-Plus (GenDEPOT). For the loading control, anti-β-tubulin antibody (Santa Cruz Biotechnology) or anti-lamin B1 antibody (Abcam) was used.

IK was isolated from the supercritical carbon dioxide (SC-CO₂) extract, a radiation-induced mutant cultivar of Perilla frutescens var. crispa [23 (link)]. Specific antibodies for p44/42 MAPK (ERK1/2), phospho (p)-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204), SPK/JNK, p-JNK (Thr183/Tyr185), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). Anti-β-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, USA).

Polarized T84 cells, apically incubated with GC for 6 h with or without inhibitors, were lysed by RIPA buffer [0.1% triton × 100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 1 mM Na₃VO₄, 50 mM NaF, 10 mM Na₂PO₄, and proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)]. Lysates were resolved using SDS-PAGE gels (BioRad, Hercules, CA) and analyzed by Western blot. Blots were stained for pMLC or MLC (Cell Signaling Technology), stripped, and reprobed with anti-β-tubulin antibody (Santa Cruz, Santa Cruz, CA). Blots were quantified using a Fujifilm LAS-3000 (Fujifilm Medical Systems U.S.A., Inc., Stamford, CT).

Atractylenolide-I (AT-I, purity > 98%) and paclitaxel (purity > 98%) were purchased from Selleck Chemicals (Houston, TX, USA). A human CTGF/CCN2 DuoSet ELISA kit and recombinant human CTGF protein (rCTGF) were purchased from R&D Systems (Minneapolis, MN, USA). AT-1 was used at a dose of 0-100 μM (17 (link)). rCTGF was used at a dose of 0-20 μg/mL (38 ). TRIzol reagent was bought from Invitrogen (Carlsbad, CA, USA). M-MLV reverse transcriptase was bought from Promega (Madison, WI, USA). SYBR Green Real-time PCR Master Mix was bought from TOYOBO (Osaka, Osaka Prefecture, Japan). Puromycin was bought from Coolaber Technologies (Beijing, China). Anti-CTGF, anti-αSMA and anti-FAP antibodies were obtained from Abcam (Cambridge, UK). Anti-β-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies were obtained from OriGene Technologies (Rockville, MD, USA). Immobilon Western Chemiluminescent HRP Substrate was bought from Millipore (Billerica, MA, USA).