Vinculin antibody

Hs578t cells were seeded on a glass coverslip in 6-well plates at 50% of confluence. Following cell attachment the medium was removed and cell were fixed with 4% Paraformaldehyde containing 0.3% Triton X100. Cells were washed three times in 1X PBS and incubated with blocking buffer (0.1% Triton X100, 3% BSA in PBS). Vinculin antibody (Sigma #V4505; dilutiom: 1/50) was used to detect the FAPs. Phospho-Myosin light chain-2 antibody (Cell Signaling Technology #3671) was used at 1/50 dilution. F-Actin was stained with rhodamine phalloidin dye (Invitrogen #R415) at 1/200 dilution. Nuclei were visualized using Hoecht 33342 dye (Invitrogen #H3570) at 10 μg/mL. Images were acquired under a Zeiss 780 confocal microscope.

For immunofluorescence experiments, cells (40% confluent) were cultured on glass slides, fixed with 4% paraformaldehyde and then washed with PBS 1X. For vinculin analysis, cells were seeded on glass slides coated with 0.5 μg of rat tail collagen I (Invitrogen) o/n at 4°C and blocked with 1% BSA for 1h30. Vinculin antibody (1/200, Sigma) diluted in PBS containing 1% BSA was used. Secondary antibody tagged with Alexa-488 (1/1000, Life Technology) and/or rhodamin-phalloidin (1/750, Sigma) were then added for 1h. Nuclei were counterstained with Hoescht staining. Immunofluorescence was analyzed using Zeiss-AxioImager or LSM780 Confocal microscope. Vinculin quantification was performed using computational analysis.

Preparation procedures and characterization data for C-6 and its analogs can be found in a supplementary file (Additional file 1). The following primary antibodies were purchased from Cell Signaling (Danvers, MA, USA): CHOP, GRP78, p-EIF2α, pan-EIF2α, p-JNK, and pan-JNK. In addition, the vinculin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Actinomycin D (ACTD) and cycloheximide (CHX) were also obtained from Sigma-Aldrich.

Whole-cell extracts were obtained upon lysis of cells in cold RIPA buffer (Thermo) with 1x concentration of PhoSTOP and Protease Inhibitor (Roche). Once protein concentration was determined using BCA assay (Thermo), 40 μg samples were boiled for 5 mins in SDS sample buffer and ran on SDS-PAGE gel. Immunoprecipitation was performed using ImmunoCruz™ IP/WB Optima B System (Santa Cruz) based on the manufacturer’s guideline. 2 µg of primary antibody, or immunoglobulin G (IgG) was used for immunoprecipitation and control respectively. Transfer was done onto PVDF membranes, blocked with Odyssey Blocking Buffer (Licor), probed with primary antibodies at 1:1000 dilution. Antibodies for STAT3, pSTAT3 S727 and Y705, PSA, c-MYC, Cyclin D1 and PARP were all obtained from Cell Signaling. Vinculin antibody was purchased from Sigma while AR and LaminB1 were purchased from Santa Cruz. Image acquisition and intensity quantifications were carried out using Licor Odyssey Scanner with Application Software V3.0 and normalized to Vinculin intensity. Full length blots are provided as Supplementary Figs S5–S9.

Cells were allowed to attach for 3 h to dishes pre-coated with Matrigel. Cells were then fixed with 4% formaldehyde in PBS, permeabilized with 0.1% Triton X-100 and blocked with 1% BSA (Beyotime, Shanghai, China) in PBS for 30 min. Dishes were incubated with the vinculin antibody (Sigma, St. Louis, MO, US) at a 1∶500 dilution for 2 h and with rhodamine-phalloidin (Molecular Probes, New York, US) at a 1∶100 dilution in PBS for 1 h. Antibody-treated cells were washed in PBS and incubated with an Alexa 594 goat anti-mouse secondary antibody (Pierce, Rockford, IL, US) at a 1∶400 dilution in PBS for 1 h. Cell nuclei were stained with DAPI (Vector Labs, CA, US) for 5 min. Cells probed with rhodamine-phalloidin were washed. Finally, cells were observed with an A1 confocal microscope (Nikon, Tokyo, Japan).

Commercial pure titanium (ASTM Grade 2, Ti) and titanium alloy (Ti6Al4V) rods were made by the Baoji titanium industry company (China). Nitric acid (HNO₃, 68%, w/w), hydrofluoric acid (HF, ≥ 40%, w/w), and saline were bought from Sichuan Kelun Pharmaceutical CO., LTD. (China). Enrofloxacin (C₁₉H₂₂FN₃O₃) was bought from Adams (China). Pentobarbital sodium (C₁₁H₁₇O₃N₂Na) was obtained from Ailu Biological Tech Co. Ltd. (China). Povidone‐iodine was from Jinshan Pharmaceutical Company (China). Calcein (C₃₀H₂₆N₂O₁₃), toluidine blue, and Giemsa stain solution were from Solarbio (China). Aluminum oxide (Al₂O₃) particles of 40 grit (0.4 mm in diameter) for sandblasting were obtained from Bioactive Material Co. Ltd. (BAM, China). Vinculin antibody, basic fuchsin, and rhodamine‐conjugated phalloidin were from Sigma (USA). FITC conjugated goat‐anti‐rabbit secondary antibody, Trizol reagent, 4', 6‐diamidino‐2‐phenylindole (DAPI), and 5‐chloromethylfluorescein diacetate (CMFDA) were from Thermo (USA). SuperScript III Reverse Transcriptase was from Invitrogen (USA). The PCR array of mouse osteogenesis‐related genes (RT² Profiler PCR Array Mouse Osteogenesis, PAMM‐026Z) was purchased from QIAGEN.