Hrp labeled secondary antibody

DMEM, PBS, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Rabbit antibodies against TLR2, cy3-conjugated secondary antibody, 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Abcam (Cambridge, UK). Rabbit antibodies against p65, p-IκBα, IκBα, p-JNK, JNK, p-ERK, ERK, p-p38, p38, mouse antibody β-actin, and HRP-labeled secondary antibodies were products of Cell Signaling Technology (Beverly, MA, USA). The ELISA assays kit for TNF-α, IL-1β, and IL-6 were bought from Thermo Fisher (Invitrogen, Waltham, MA, USA). The pleuropneumonialike organisms (PPLO) broth was obtained from BD Biosciences (Franklin Lake, NJ, USA). Triton X-114 was product of Solarbio (Beijing, China). HRESIMS were measured using AB SCIEX Triple TOF 6600 mass instruments (SCIEX, Framingham, MA, USA). UPLC was performed on Agilent 1290 (Agilent, Palo Alto, CA, USA). Silica gel (200–300 mesh, Qingdao, China), HP-20 macroporous resin (Lupital, Japan) were utilized for column chromatography. Subsequently, ODS column chromatography was applied to obtain compound 1 and compound 3 in TFCO.

Western blots were performed as previously described [17 (link)]. Primary antibodies against ACTB (Santa, TX, USA), TRPV4 (alomone labs, Israel), ZEB1, E-cadherin, N-cadherin and Vimentin (Cell Signaling Technology, MA, USA) were incubated at 4 °C overnight with constant shaking. HRP labeled secondary antibodies (Cell Signaling Technology, MA, USA) were incubated at room temperature for 1 h.

Drugs were added to cocultures of MDA-MB-231 and MC3T3-E1 cells for 24 h as described above. Total protein was extracted from the cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China). Equal amounts of protein extracts were then separated using 10% sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Gibco, Grand Island, USA) and transferred onto a polyvinylidene fluoride (PVDF) membrane (0.45 μm) (Millipore, Bedford, MA, USA). The membranes were blocked with 5% w/v nonfat dry milk (Gibco, Grand Island, USA) dissolved in Tris-buffered saline (Amresco, Solon, OH, USA) plus Tween-20 (TBS-T) (Gibco, Grand Island, USA) at 25°C for 1 h, followed by overnight incubation with primary antibodies at 4°C. The primary antibodies for immunoblotting were anti-OPG antibody (P-17) (sc-21038, Santa Cruz, CA, USA), anti-RANKL antibody (ab124797, Abcam, Cambridge, UK), and anti-β-actin antibody (N-21) (sc-130657, Santa Cruz, USA). After washing with TBS-T (CoWin Biotech, Beijing, China), the membranes were incubated with HRP-labeled secondary antibodies (Cell Signaling Technologies, Danvers, MA, USA) for 1 h at 25°C. The membranes were analyzed using a protein visualizer ECL (Tanon Science and Technology, Shanghai, China).

Related primary pancreatic stellate cells were harvested by RIPA lysis buffer containing 1 mmol/l PMSF and centrifuged at 12000 rpm for 10 min at 4 °C. All protein sample concentration was determined by the BCA method (Thermo, Carlsbad, CA, USA). The proteins were separated by 10% or 15% of SDS/polyacrylamide gels, which were transferred to the PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% milk for 1 h at RT, then incubated with primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST for three times, incubated with HRP-labeled secondary antibodies (Cell Signaling, Beverly, MA, USA). The ECL reagents (Thermo, Carlsbad, CA, USA)were added to visualize the chemiluminescence by ECL Plus detection system (Tanon, Shanghai, China). The band densities were analyzed with the ImageJ analysis system.

Cells were lysed with modified lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, and 0.5% deoxycholate). The BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA) was used to quantify protein concentrations. Samples containing approximately 40 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA), transferred to PVDF membranes (Millipore, Billerica, MA, USA), then incubated with primary antibodies against NAT10 (1 : 1000, Santa Cruz), Twist (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), vimentin (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1 : 1000, Cell Signaling Technology), and GAPDH (1 : 1000, Cell Signaling Technology), followed by HRP-labeled secondary antibodies (1 : 2000, Cell Signaling Technology), then the bands were detected using chemiluminescent reagent (GE Healthcare, Piscataway, NJ, USA). The optical density of each band was quantified and expressed relative to the internal control GAPDH.

Optic nerves were collected into RIPA buffer (PBS, 0,5% deoxycholate, 0,1% SDS, 1% NP40) containing protease (1%) and phosphatase (1%) inhibitors (Sigma), in presence of 2 stainless steel beads in order to be homogenized with TissueLyser system. Three micrograms of proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked in 5% skimmed milk during 2 hours, followed by overnight incubation at 4 °C with anti-MAG antibodies or β-actin (Abcam, France). After three washes with TBS-0,1%Tween20, the membrane was incubated with the corresponding HRP-labeled secondary antibodies (Cell Signaling). Then, the membrane was observed using ImageQuant LAS 4000 (GE Healthcare).