α tubulin 2125

Cells treated with physciosporin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [30 (link)]. Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), and Msi-1 (ab52865, Abcam) were used to detect stemness factors. α-tubulin (2125, Cell Signaling Technology) antibody was used as an internal control. The luminescence photon from proteins were detected by an Image Quant LAS 4000 mini using horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Quantitation of bands were performed by using Multi-Gauge 3.0 software, and the relative density of each band was calculated based on the density of the internal control bands in each sample. Values are shown as arbitrary densitometric units corresponding to signal intensity. All results are representative of at least three independent experiments.

Recombinant Human and murine TNFα were obtained from Peprotech. LPS (from Escherichia coli O111:B4) was purchased from Sigma. VCAM-1 (sc-13160), ICAM-1 (sc-1511-R) and β-actin (sc-47778) antibodies were from Santa Cruz Biotechnology. TRIM47 antibody (26885-1-AP) was purchased from Proteintech. Phospho-p65 (3033), p65 (8242), IκBα (4812), phospho-IκBα (2859), phospho-IKKα/β (2078), IKKα (11930), IKKβ (8943), phospho-JNK (4668), JNK (9252), phospho-ERK1/2 (4370), ERK1/2 (4695), phospho-p38 (4511), p38 (8690),TRAF2 (4724), TRAF6 (8028), TAK1(5206), IKKγ (2685), TBP (44059), Flag (8146 and 2368) and α-tubulin (2125) antibodies were purchased from Cell Signaling Technology. VE-cadherin (ab33168), ubiquitin (ab7780), ubiquitin (K48, ab140601), and ubiquitin (K63, ab179434) antibodies were purchased from Abcam.

Aprotinin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride (NaF), propidium iodide, sodium orthovanadate, mitomycin C, type I collagen, and tetrahydrofuran (THF) were purchased from Sigma Chemical Co. (St Louis, MO). Antibodies (Abs) against AQP-3 (sc-20811), p38 (sc-535), ERK2 (sc-154), JNK1/3 (sc-474) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Abs against p-AKT (#9271), Akt (#9272), p-ERK1/2 (#9101), p-P38 (#9211), p-JNK (#9251), and α-tubulin (#2125) were obtained from Cell Signaling Technology (Beverely, MA). U0126, MK2206, and SB203580 were obtained from Merck Millipore (Billerica, MA). The 18β-GA derivative (18β-GA-d) was provided by Dr. Chun-Nan Lin. For all the experiments in the study, the 18β-GA-d was dissolved in THF.