Irdye 800cw conjugated goat anti rabbit igg

HTM cells (N17TM-2, N27TM-2 and N27TM-6) were plated into 96 well plates at 15,000 cells per well. One week after the cells reached confluency media was replaced with media containing 1% FBS and 2ng/ml TGFβ2 with and without 2μM FUD. Two days later the cells were processed for OCW analysis as previously described [19 (link)]. Briefly, cells were extracted with a hypotonic buffer (20mM HEPES, pH 7.4, 1mM EDTA, 1% sodium deoxycholate and HALT^TM protease inhibitor (Thermo Fisher Scientific)) leaving behind insoluble extracellular matrix (ECM). The DOC insoluble ECM was fixed with 4% paraformaldehyde and the total protein in the wells was labeled with IRDye 680 NHS ester (LI-COR). Wells were then blocked and fibronectin was detected using rabbit anti-fibronectin sera followed by the IRDye 800CW-conjugated goat anti-rabbit IgG (LI-COR). The plates were then read on a LI-COR Odyssey CLx scanner and analyzed using the LI-COR Image Studio v. 5.0.21 software. The antibody signal was normalized to the corresponding NHS ester signal (total protein).

NSCLC cells were seeded into 6-well plates (0.15–0.45 × 10⁶ cells per well, depending on the doubling time of the different cell lines). After 1 day (i.e. day 0), 1 μM MTA or concomitant chemotherapy treatment was used to treat cells. On day 2, 10 μM cisplatin was added for schedule chemotherapy treatment, e.g. 48 h of MTA pretreatment followed by 24 h of combined MTA and cisplatin treatment. Cells were lysed by RIPA buffer containing 1× protease and phosphatase inhibitor cocktail at different timepoints. Protein concentration was measured by Pierce™ BCA Protein Assay Kit. Equal amounts of protein lysates (11–23 μg/ lane) were resolved by SDS-PAGE. Then the bands were transferred onto nitrocellulose membranes. The membrane was firstly blocked in Intercept® (TBS) Blocking Buffer for 1 h at room temperature and then blotted with specific primary antibodies (Table S1, Additional file 3) at 4 °C overnight with shaking. Tris Buffered Saline (TBS) with 0.2% Tween-20 was used to wash the membrane. IRDye 680LT-conjugated goat anti-mouse IgG and IRDye 800CW-conjugated goat anti-rabbit IgG from Li-COR Biosciences were used at 1:5000 dilutions. Finally, signals of membrane-bound secondary antibodies were imaged using the Image Studio Lite System, also for image analysis.

Bladder cancer cell line RT112 and KU7 were used in this study and cultured as described previously[14 (link),20 (link)]. RT112 and KU7 cell lines were purchased from cell bank of Chinese Academy of Science (Shanghai, China). RT112 and KU7 cells were mainly used in the following study, which originate from non-invasive bladder cancer. The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated at 37°C with a humidified 5% CO² atmosphere.
The Eg5 inhibitors, S(MeO)TLC were purchased from Bachem (Bubendorf, Switzerland) and were dissolved in dimethyl sulfoxide (DMSO). Antibody anti-RRM1 and anti-RRM2 (Rabbit) was purchased from Abcam (USA); IRDye 800CW conjugated goat anti-rabbit IgG were from Li-Cor Biosciences (USA). Prime Script RT-PCR kit was purchased from TaKaRa (China). TRIzol was purchased from Invitrogen (USA).
140 cases of bladder cancer tissues were collected from Shandong Provincial Hospital Affiliated to Shandong University from December 2005 to March 2007. Radiotherapy, chemotherapy and immunotherapy were not performed before the surgery and all samples were verified by two pathologies after the surgery.

Proteins were separated by 10% (wt/vol) SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with primary antibodies (all at 1:1,000 dilution) overnight at 4 °C, and subsequently incubated with IRDye 800CW-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (both at 1:10,000 dilution; LI-COR Biosciences, Lincoln, NE, USA) for 1 h. Protein bands were visualized with Odyssey infrared imaging system (LI-COR Biosciences).